Methods for expression and purification of immunotoxins

ABSTRACT

The present invention relates to a method of expressing an immunotoxin in  Pichia pastoris  strain mutated to toxin resistance comprising a) growing the  Pichia pastoris  in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaining a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/IO L of initial volume during induction along with a continuous infusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h.

This application claims benefit of U.S. Provisional Application60/491,923 filed Aug. 1, 2003. The U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods of protein expressionand purification, and more specifically, to methods of expression andpurification of immunotoxins.

2. Description of the Related Art

The number of organ transplants performed in the United States each yearis approximately 24,000 and consists predominantly of kidney transplants(14,000), liver transplants (5,000), heart transplants (2,200), andsmaller numbers of pancreas, lung, heart-lung, and intestinaltransplants (2002 OPTN/SRTR Annual Report).

Transplant tolerance remains an elusive goal for patients and physicianswhose ideal would be to see a successful, allogenic organ transplantperformed without the need for indefinite, non-specific maintenanceimmunosuppressive drugs and their attendant side effects. Many of thesepatients have been treated with cyclosporin, azathioprine, andprednisone with a variety of other immunosuppressive agents being usedfor induction or maintenance immunosuppression. The average annual costof maintenance immunosuppressive therapy in the United States isapproximately $11,000 (Immunosuppressive Drugs Coverage Act, NationalKidney Foundation, available athttp://www.kidney.org/general/pubpol/immufact.cfm). While these agentsare effective in preventing rejection, the side effects ofimmunosuppressive therapy are considerable. Immunosuppressive therapyinduces nonspecific unresponsiveness of the immune system. Recipientsare susceptible to infection and there is a risk of malignancy such asin the form of post transplant lymphoproliferative disorders. A majorgoal in transplant immunobiology is the development of specificimmunologic tolerance to organ transplants with the potential of freeingpatients from the side effects of continuous pharmacologicimmunosuppression and its attendant complications and costs.

A bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G₄S) was developedfor tolerance induction for transplantation, T-cell leukemia andautoimmune diseases. The immunotoxin consists of the first 390 aminoacid residues of diphtheria toxin (DT390) and two tandem antigen-bindingdomains (sFv) from the anti-CD3 antibody UCHT1, that are responsible forbinding the immunotoxin to the CDR3εγ subunit of the T cell receptorcomplex. The anti-CD3ε antibody moiety enables the immunotoxin to targetspecific cells and the diphtheria toxin moiety kills the target cells.The immunotoxin may be utilized to effect at least partial T-celldepletion in order to treat or prevent T-cell mediated diseases orconditions of the immune system.

Administration of an anti-T cell immunotoxin provides an approach forspecific immunologic tolerance. It is applicable to new organtransplants and potentially to existing transplants in recipients withstable transplant function. The immunotoxin can provide highly specificimmunosuppression and imparts transplant tolerance in primates, withoutthe adverse effects of nonspecific immunosuppressive drugs,anti-lymphocyte serum or radiation. It is a goal in this field toinhibit the rejection response to the point that rejection is not afactor in reducing average life span among transplant recipients.

The methylotrophic yeast Pichia pastoris has been used successfully toexpress heterologous proteins from different origins (Gellissen 2000).As an eukaryote, Pichia pastoris has the ability to perform manypost-translational protein modifications such as proteolytic processing,folding, disulfide bond formation and glycosylation. Like other yeasts,Pichia pastoris offers significant advantages over higher eukaryoticcells such as Chinese hamster ovary (CHO) or baculovirus-infected insectcell expression systems. It is easy to manipulate, has a rapid growthrate and requires inexpensive media. These greatly reduce the productiontime and cost, especially on a commercial scale. Unlike Saccharomycescerevisiae, Pichia pastoris is not a strong fermentor and can be easilycultured to very high cell density of >100 g dry cell weight/liter(Siegel et al., 1989). This, plus the strong AOX1 promoter employed indriving transcription of foreign genes, have made Pichia pastoris thesystem of choice for high levels of expression of heterologous proteins.The AOX1 promoter also has advantages in the expression of foreignproteins that are deleterious to the expressing host because thepromoter is tightly regulated and highly repressed under non-methanolicgrowth conditions. The inducible and tightly regulated AOX1 promoter hasallowed successful expression of DT based immunotoxins, in secretedform, in Pichia pastoris strains without any mutation to confer aresistance to DT. (Woo et al., 2002). However, diphtheria toxin (DT) isa very potent toxin to all eukaryotic cells if its catalytic domain canfind a route to the cytosol. Pichia pastoris is inherently sensitive tothese toxins.

The prior art teaches methods for growing Pichia pastoris. For example,Pichia pastoris may be grown in a fermentor. One protocol for Pichiapastoris fermentation contains glycerol as the initial carbon source,followed by brief carbon starvation and use of methanol as the carbonsource (Pichia pastoris Fermentation Using a BioFlo 110 BenchtopFermentor, New Brunswick Scientific).

Woo et al. disclosed that, when expressing a bivalent anti-human anti-Tcell immunotoxin A-dmDT390-bisFv(G₄S) in Pichia pastoris, a bufferedcomplex medium at pH 7.0 with 1% casamino acids provided the highestexpression in shake flask culture and that the expression level wasimproved by adding PMSF in the range of 1 to 3 mM. (25 ProteinExpression and Purification 270-82 (2002)).

Sreekrishna disclosed that an increased secretion level was obtainedusing Pichia pastoris in shake flask cultures when the cells were highlyaerated and in a buffered medium at pH 6.0 that was supplemented withyeast extract and peptone (Chapter 16, Industrial Microorganisms: Basicand Applied Molecular Genetics (1993)). The growth medium containedyeast nitrogen base with ammonium sulfate, biotin and glycerol bufferedto pH 6.0 with potassium phosphate buffer as well as yeast extract andpeptone. The induction medium contained methanol in place of glycerol.

In contrast, the present invention provides an improved method of usingPichia pastoris to produce an immunotoxin. The immunotoxins expressedand purified in the present invention can be used in a method ofinducing immune tolerance. It would be desirable to provide a method ofexpression and purification that increased the yield of immunotoxins.The present invention addresses this problem and others in the mannerdescribed below.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a method of expressing animmunotoxin in Pichia pastoris toxin resistant EF-2 mutant comprising a)growing the Pichia pastoris in a growth medium comprising an enzymaticdigest of protein and yeast extract; and b) performing methanolinduction of the Pichia pastoris with a limited methanol feeding of 0.5to 0.75 ml/min (per 10 L initial medium) during induction, and whereinthe methanol induction is at a temperature of below about 17.5° C.

In another aspect, the present invention relates to a method ofexpressing an immunotoxin in Pichia pastoris comprising a) growing thePichia pastoris in a growth medium comprising an enzymatic digest ofprotein and yeast extract; and b) performing methanol induction of thePichia pastoris with a methanol and glycerol containing feed, whereinthe Pichia pastoris is contacted with a phenylmethanesulfonyl fluorideand a source of amino acids and wherein the methanol induction is at atemperature of below about 17.5° C.

In yet another aspect, the present invention relates to a method ofpurifying a non-glycosylated immunotoxin comprising a) loading asolution containing the non-glycosylated immunotoxin onto a hydrophobicinteraction column; b) obtaining a first non-glycosylated immunotoxincontaining eluant from the hydrophobic interaction column; c) loadingthe non-glycosylated immunotoxin containing eluant from step (b) onto ananion exchange column; d) obtaining a second non-glycosylatedimmunotoxin containing eluant from the anion exchange column by elutingthe non-glycosylated immunotoxin with a sodium borate solution; e)diluting the concentration of sodium borate in the secondnon-glycosylated immunotoxin containing eluant from step (d) to about 50mM or less; f) concentrating the diluted non-glycosylated immunotoxincontaining eluant from step (e) over an anion exchange column; and g)obtaining a purified non-glycosylated immunotoxin from the anionexchange column.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention.

FIG. 1. (a) Conservation of diphthamide domain and DT-resistantmutations in eukaryotic EF-2s. (b) Nucleotide sequence mutations for thesubstitution of Arg for Gly 701 in Pichia pastoris EF-2. The underlinedsequences are the site for the restriction enzyme Sac II that resultedfrom the nucleotide mutations. See SEQ ID NOS: 140.

FIG. 2. The 5′ end sequence of Pichia pastoris EF-2 showing the shortintron (SEQ ID NO: 11; mRNA) and (SEQ ID NO: 12; gDNA). The 5′ splicesite, branch site and 3′ splice site are under lined. EF-2 codingsequence is in bold.

FIG. 3. (A) (B) (C) (D) Nucleotide and deduced amino acid sequence ofPichia pastoris EF-2 (SEQ ID NO:13). The nucleotide sequence is numberedfrom the beginning of the initiation codon. Consensus GTP-binding motifin the protein sequence is AHVDHGKST (SEQ ID NO:14), the threonineresidue putatively phosphorylated in vivo by EF-2 kinase is circled andthe effector domain conserved among all elongation factors isDEQERGITIKSTA (SEQ ID NO:15). The 22 well-conserved residues of thediphthamide domain are boxed.

FIG. 4. Targeted mutation using the 3′ sequence of EF-2 that has beenmutated in vitro. The mutating plasmid pBLURA-Δ5′ mutEF-2 contains fouressential elements: β-lactamase gene(Ampr), Uracil selection marker(URA3), 3′AOX1 transcription termination sequence(TT) and the in vitromutated FF-2 3′ sequence, Δ5′ mutEF-2.

FIG. 5. Agarose gel electrophoresis of PCR products of selected Ura+clone derived from Pichia pastoris JC308 strain. (a) PCR products withprimers 1 and M; (b) PCR products with primers 2 and w; (c).Sca IIdigested PCR products with primers 2 and 3.

FIG. 6. Western blot analysis of cytosolic expression of DT-A chain inmutated and wild type Pichia pastoris strains. (a) Lanes 1-6 are thecell extracts of 6 independent clones of mutEF2JC307-8 transformed withpPIC3-DtA, +C: The purified A-dmDT390-bisFv. M: SeeBlue plus2 Proteinmarkers (Invitrogen). (b) Cytosolic expression of DT-A chain in culturesof two separated colonies of mut-3 and mut-5 that are mutEF2JC307-8(3)and (5) respectively, C3 and C4. Protein samples are loaded on 4-12%NuPAGE gels (Invitrogen).

FIG. 7. The effect of intra-cellular expression of DT-A on the survivalof Pichia pastoris strains with mutated or wild type EF-2. Mut-3 and Mut5 are EF-2 mutants mutEF2JC307-8-DtA(3) and (5) respectively, Mut-3expressed DT A chain in the cytosol, mut-5 did not. C3 and C4 are thewild type EF-2 strains that did (C4) or did not express DT A chain inthe cytosol. The first bar in each category indicates the colony-formingunits before methanol induction. The second bar in each categoryrepresents the colony-forming units after methanol induction.

FIG. 8. Schematic presentation of plasmid construction. (a).pBLARG-A-dmDT390-bisFv; (b). pPGAPArg-A-dmDT390-bisFv; (c).pPGAPHis-A-dmDT390-bisFv.

FIG. 9. Western blot analysis of expression of A-dmDT390-bisFv. Samplesof culture media (a) and cell extracts (b) were loaded on 4-12% NuPAGEgels (Invitrogen). Lanes of +c are purified A-dmDT390-bisFv. Lanes 1-9were samples of 9 selected clones of mut EF2JC303 transformed with 2copies of the A-dmDT390-bisFv gene. Lanes 10, 11 and 12 were samples ofsingle copy clones: lane 12 was the non-mutated EF-2 clone JHW#2, lanes10 and 11 were two of selected clones of mutEF2JC307-8(1) andmutEF2JC307-8(2) that is also called YYL#8-2.

FIG. 10. Comparison of the methanol consumption rate among differentPichia pastoris strains. All of these strains are Mut+ (Methanolutilization plus) except for pJHW#3, which is MutS (Methanol utilizationslow). pJHW#2 to 5 and the EF-2 mutant YYL#8-2 all expressed thebivalent immunotoxin A-dmDT390-bisFv. X-33 is a wild type strain thatdoes not express A-dmDT390-bisFv, but was transformed with theexpression vector.

FIG. 11. Comparison of profiles of methanol consumption rate betweenX-33 and JW102 and between different nutrient feeding of JW102 at theindicated temperature. YE and casa represents feeding of yeast extractand casamino acids, respectively.

FIG. 12 Lowering agitation speed in fermentation reduces immunotoxinaggregates. Fermentation performed at high agitation speed resulted inmore than 50% of the secreted immunotoxin being present in inactiveaggregate forms in the supernatants. In addition, aggregates accumulatedover induction time. However, lowering agitation speed from 800 rpm to400 rpm reduced immunotoxin aggregates. Immunotoxin aggregates weremaintained at the same level over induction time.

FIG. 13 Effect of TWEEN 20® on aggregation of purified immunotoxin after20 hrs incubation at 30° C. at 250 rpm. Using purified immunotoxin,TWEEN 20® prevented the formation of aggregates by agitation.Approximately 50% of the purified immunotoxin was aggregated byincubation at 30° C. at 250 rpm for 20 hours. However, 0.01%-0.04% ofTWEEN 20® significantly reduced the aggregation purified immunotoxin byagitation.

FIG. 14. Change of gain of wet cell density during the first 44 hours ofmethanol induction. MeOH, methanol alone and feeding of casamino acids;M:G=4:1, methanol/glycerol mixed feeding and feeding of casamino acids;YE+MeOH, feeding of yeast extract and methanol alone; YE+4:1, feeding ofyeast extract and methanol/glycerol mixed feeding.

FIG. 15. Expression level of the bivalent immunotoxin and its finalpurification yield depending on induction temperature. A: change ofexpression level by induction temperature. B: change of the finalpurification yield from 1 liter of supernatant taken at 22, 44, and 67hours of methanol induction. 22 hrs, 44 hrs, and 67 hrs represent timeof methanol induction. C: change of methanol consumption depending oninduction temperature.

FIG. 16. A representative of optimized fermentation runs. Samples takenat indicated induction time points were fractionated on 4-20%SDS-tris-glycine gel and the gel was stained with Coomassie blue dye.Arrow indicates the position of the bivalent immunotoxin. Mark 12 marker(Invitrogen) was used.

FIG. 17. SDS-PAGE analysis of proteins obtained by butyl 650M capturestep. Lane 1˜4, sample flow-through fraction #1˜#4; lane 5, pooledsample flow-through fractions; lane 6˜8, wash fraction #1˜#3; lane 9,pooled wash fractions; lane 10, 11, 17, supernatant; lane 12, Mark 12protein standards (Invitrogen); lane 13˜15, eluted fraction #1˜#3; lane16, pooled eluted fractions. IT, immunotoxin.

FIG. 18. SDS-PAGE analysis of proteins obtained by Poros 50 HQ borateanion exchange step. Lane 1, Mark 12 protein standards (Invitrogen);lane 2, sample obtained from Butyl 650M HIC step; lane 3˜7, sampleflow-through fraction #1—#5; lane 8, fraction #1 eluted with 25 mMborate in Buffer B; lane 9, fraction #2 eluted with 50 mM borate inBuffer B; lane 10, fraction #3 eluted with 75 mM borate in Buffer B;lane 11, fraction #4 eluted with 100 mM borate in Buffer B; lane 12,fraction #5 fraction eluted with 1 M NaCl in Buffer B. IT, immunotoxin.

FIG. 19. Analytical gel filtration and SDS-PAGE analysis of purifiedimmunotoxin. A: Chromatogram of Superdex 200 10/300 GL gel filtration.B: Picture of Coomassie-stained SDS-polyacrylamide gel.

FIG. 20. (a) (b) (c) Amino acid sequence of Ala-dmDT390bisFv(UCHT1) (SEQID NO:16).

FIG. 21. Comparison of profiles of cell growth, methanol consumption andimmunotoxin secretion during methanol induction. Panel A. X-33 strainand the immunotoxin producing toxin resistant EF-2 mutantmutEF2JC307-8(2). These two strains had similar profile of methanolconsumption rate and wet cell density gain during methanol induction.The data shown in panel A was for X-33. For the toxin resistant mutant,the maximum methanol consumption rate and wet cell density gain at 44 hof methanol induction was 2.2 ml/rain and 9.17%, respectively. PanelsB-F. strain JW102. Constant conditions in all panels A-F were a glycerolbatch phase followed by a glycerol-fed batch phase prior to induction.For induction, either pure methanol (MeOH) alone or 4:1methanol:glycerol (M/G) mixed feed was used. PMSF at 10 mM in methanolwas infused continuously during induction. Casamino acid feeding wasperformed when yeast extract (YE) feeding was not done. Inductionconditions: panel A, M/G feeding and no YE feeding; panel B, methanolfeeding and no YE feeding; panel C, methanol feeding and YE feeding;panel D, M/G feeding and YE feeding; panel E, M/G feeding and no YEfeeding; panel F, M/G feeding and YE feeding. The induction temperaturewas 23˜25° C. in A-E and 15° C. in F (note the right hand axis in panelF is compressed 2-fold compared to the other panels). Methanolconsumption rate (ml/min), dotted line; wet cell density (%, w/v), solidline; and level of secreted immunotoxin (mg/L), dashed line. Because ofthe large amount of work involved in 10 L bioreactor fermentations, itwas not practical to replicate the results in panels A-E. The optimizedmethod, panel F, was performed 3 times and the points are averages withstandard error of the mean shown when greater than 10%. The actual datapoints for wet cell density and level of secreted immunotoxin are shownas squares and circles. The actual data points for methanol consumptionrate are omitted because methanol consumption rate was measured everyminute.

FIG. 22. Protein degradation and immunotoxin production. A. Time courseof immunotoxin levels during methanol induction in cultures withmethanol and yeast extract feeding (FIG. 21C). The immunotoxin band ismarked with IT and an arrow. B. Analysis of residual immunotoxin bySDS-PAGE after incubation (28° C., 20 h, and 250 rpm shaking) of anequal volume of purified immunotoxin (250 μg/ml) with an equal volume ofthe supernatants collected at the indicated times following methanolinduction. Mixtures of equal volumes of purified immunotoxin and PBSbuffer or supernatant from 0 h, were used as the controls (CON). Ten μlof the prepared samples were loaded for SDS-PAGE and fractionated on4˜20% SDS-tri-glycine gels under non-reducing conditions. Gels werestained with Coomassie blue dye. Mark12 marker (Invitrogen) was used asthe protein marker.

FIG. 23. Effect of temperature on immunotoxin production. Samples takenat indicated induction time points (44, 50, 67 h) from runs at differentinduction temperature (15˜23° C.) were fractionated on 4˜20%SDS-tris-glycine gels under non-reducing condition. Continuous feedingof yeast extract and methanol-glycerol feed were used for all runs. Thegels were stained with Coomassie blue dye. IT-dp-degraded products ofthe bivalent immunotoxin. These degradation products were identified byWestern blots using anti-DT antibody and anti-(G₄S)₃ linker antibody.The anti-(G₄S)₃ linker antibody could detect the bivalent immunotoxinand degraded products, because the immunotoxin contained three (G₄S)₃linkers. Arrows point to bands not related to the bivalent immunotoxin.IT—bivalent immunotoxin. Mark12 marker (Invitrogen) was used as theprotein marker.

FIG. 24. Analysis of protease activity in supernatants in the absenceand presence of PMSF during methanol induction at 15° C. Supernatantswere taken at 0, 22, 44 and 67 h of methanol induction from fermentationruns treated with continuous feeding of yeast extract andmethanol-glycerol feed. The supernatants were incubated with unnickedCRM9 as the substrate. After incubation, 10 μl of the sample wasfractionated on 4˜20% SDS-tris-glycine gels under reducing conditions.After staining and drying, the gel was digitized and analyzed for bandintensity of =licked CRM9 by using NIH Image software. PMSFsupplementation during methanol induction, solid line; no PMSF, dottedline. Each data point is the average from 3 fermentation runs with thestandard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used in the specification and the appended claims, the singular aims“a,” “an” and “the” include the plural forms unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Contacted” means one substance is placed in physical association withanother substance.

“Non-glycosylated” means in the absence of glycosylation or in theabsence of glycosylation perceptible using routine methods known in theart for measuring glycosylation. Thus non-glycosylated includes havinghad glycosylation sites mutated so that glycosylation does not occur,expressing in a system in which glycosylation will not occur or notpossessing glyscosylation sites in the wild type state.

“Loading” a column means placing the sample in a position in which atleast a portion of the sample will eventually enter the part of thecolumn occupied by the resin.

An “enzymatic digest” refers to hydrolysis of a protein or peptide atpeptide bonds by one or more of various enzymes. Such enzymes mayinclude but are not limited to trypsin, chymotrypsin, pepsin, thrombin,papain, bromelain, thermolysin, subtilisin or carboxypeptidase A.

“Yeast extract” is a preparation of peptides and amino acids obtained byproteolysis of the proteins within yeast cells.

“Induction” refers to providing a signal to a given promoter to causeexpression of a given gene.

“Feed” refers to providing fresh media or nutrients at a rate that atleast partially replaces the media or nutrients as they are depleted.

“Moiety” refers to one portion of a molecule or compound that is dividedinto multiple portions. In the present invention, moiety may refer atoxin portion or an antibody portion of an immunotoxin.

Throughout this application, the term “bivalent” is used to refer to theability of a single composition to bind two ligands. For example, anA-dmDT390-bisFv(G₄S) immunotoxin can bind two CD3 molecules. It is alsounderstood that the term “divalent” can have a similar meaning in theart. Herein, the terms “bivalent” and “divalent” refer to the sameproperty and are used interchangeably.

The invention provides a system for expressing and purifying mutant ADPribosylating toxins and toxin fusion proteins in a Pichia pastorismutant. The methods of the present invention possess the advantage ofbeing compliant with Good Manufacturing Practices.

As used throughout, optionally, the immunotoxin is a fusion protein. Theimmunotoxin can comprise a diphtheria toxin moiety. It is understood andherein contemplated that other ADP ribosylating immunotoxins may be usedin the present methods. For example, specifically contemplated arefusion proteins wherein the immunotoxin comprises a Psuedomonas exotoxinA moiety. The toxin moiety can be a truncated moiety and/or can comprisemutations as compared to the wild-type toxin.

The immunotoxin can further comprise a CD3 antibody moiety or otherantibody moiety. It is also understood that the immunotoxin can comprisea targeting antibody moiety other than the CD3 antibody. One of skill inthe art would know which moiety to use with the immunotoxin based on thetarget cell. For example, a CD22 antibody may be used to direct theimmunotoxin fusion protein to B cells.

The invention provides a method of expressing an immunotoxin in Pichiapastoris toxin resistant EF-2 mutant comprising growing the Pichiapastoris in a growth medium comprising an enzymatic digest of proteinand yeast extract; and performing methanol induction at a temperature ofbelow about 17.5° C.

In one aspect, the invention provides a method of expressing animmunotoxin in Pichia pastoris toxin resistant EF-2 mutant comprisinggrowing the Pichia pastoris in a growth medium comprising an enzymaticdigest of protein and yeast extract; and performing methanol inductionof the Pichia pastoris with a limited methanol feeding during inductionof 0.5 to 0.75 ml/min (per 10 L initial medium), wherein the methanolinduction is at a temperature of below about 17.5° C.

Alternatively, the invention provides a method of expressing animmunotoxin in Pichia pastoris comprising growing the Pichia pastoris ina growth medium comprising an enzymatic digest of protein (e.g., soyprotein) and yeast extract; and performing methanol induction of thePichia pastoris with a methanol and glycerol containing feed (e.g., witha methanol to glycerol ration of about 4:1), wherein the Pichia pastorisis contacted with a phenylmethanesulfonyl fluoride and a source of aminoacids (e.g., a yeast extract) and wherein the methanol induction is at atemperature of below about 17.5° C.

The act of contacting Pichia pastoris with phenylmethanesulfonylfluoride and the source of amino acids in the expression method includescontacting the cells with phenylmethanesulfonyl fluoride and the sourceof amino acids for at least 2 hours, including 2, 3, 4, 5, 6, 7, 8, 9,10, or more hours or any amount in between. Preferably, thephenylmethanesulfonyl fluoride is dissolved in the 4:1 methanol glycerolinduction feed and the concentration does not exceed 10 mM.

The methanol induction temperature is preferably below about 17.5, andeven more preferably is about 15° C. Other temperatures at which methaneinduction can take place in the practice of the present method include17.0, 16.5, 16.0, 15.5, 14.5, 14.0, 13.5, 13, 12.5, 12° C. or anyamounts in between.

Growth medium refers to any substance required for growth of theselected organism. Substances required for growth may include but arenot limited to carbon, hydrogen, oxygen, nitrogen, phosphorus, sulphur,potassium, magnesium, calcium, sodium, iron, trace elements and organicgrowth factors. Various materials may be included in growth medium toprovide the required substances. Such substances include but are notlimited to simple sugars, extracts such as peptone, soytone, tryptone,yeast extract, carbon dioxide, vitamins, amino acids, purines andpyrimidines. An example of the method of the invention utilizes thepresence of an enzymatice digest of soy produced by DIFCO. Anotherexample of the method of the invention utilizes the presence of yeastextract produced by DIFCO. It is understood that yeast extracts andenzymatic digests produced by any manufacturer of such items, forexample, New England Biosciences can be used.

In a specific non-limiting example of the method, the composition of thegrowth medium is about 4% glycerol, about 2% yeast extract, about 2%enzymatic digest of soy protein, about 1.34% yeast nitrogen base withammonium sulfate and without amino acids, and about 0.43% PTM1 solution.Optionally, the growth medium further comprises an antifoaming agent.More specifically, the antifoaming agent is present at a concentrationof about 0.01% or greater. For example, the anti foaming agent can bepresent at a concentration of 0.07% or any amount between about 0.01%and about 0.07%. The optimum level of antifoaming reagent is chosen asthe minimum amount required to reduce the layer of foam above theliquid-air interface to ½ inch or less. Thus, the composition of thegrowth medium can be about 4% glycerol, about 2% yeast extract, about 2%enzymatic digest of soy protein, about 1.34% yeast nitrogen base withammonium sulfate and without amino acids, about 0.43% PTM1 solution andabout 0.02% antifoaming agent.

It is understood that one of skill in the art will know that thecomposition of the growth medium may be altered to optimize for maximalgrowth. Specifically contemplated are changes up to 20% above or belowthe percentages of the components in the growth medium. Thus hereindisclosed is a growth medium, wherein the composition of the growthmedium is about 3.2%-4.8% glycerol, about 1.6%-2.4% yeast extract, about1.6-2.4% enzymatic digest of soy protein, about 1.07-1.61% yeastnitrogen base with ammonium sulfate and without amino acids, and about0.34%-0.52% PTM1 solution. For example, specifically disclosed is agrowth medium, wherein the composition of the growth medium is about3.6% glycerol, about 2.4% yeast extract, about 1.9% enzymatic digest ofsoy protein, about 1.43% yeast nitrogen base with ammonium sulfate andwithout amino acids, and about 0.43% PTM1 solution.

Optionally, the dissolved oxygen concentration in the growth medium ismaintained at a value of 40% or higher (e.g., 45%, 50%, 55%, 60%, or65%) in the expression method of the invention. For example, in thepresent invention, a glycerol-fed batch phase is employed to obtain highcell density before initiation of methanol induction. The glycerol-fedbatch phase is started when the dissolved oxygen rises above 40%.Glycerol is fed whenever the dissolved oxygen rises above 40% and untilthe level drops below 40%. When the dissolved oxygen rises again afterstopping glycerol feeding, the feed is switched to methanol. A rise orspike in dissolved oxygen, DO, level indicates exhaustion of the carbonsource. Typically the DO spike is used to indicate depletion of theglycerol used for growth and indicates that a switch to methanol for theinduction phase should occur.

Furthermore, the growth step is optionally at a pH of about 3.0-4.0 andthe methanol induction step is at a pH of about 6.7-7.4. For example,the growth step is at a pH of 3.5 and the methanol induction step is ata pH of 7.0.

Methanol induction time can be increased to maximize yields. Typicalinduction times include 22 h, 44 h, 67 h, and 163 h. Inductuion can beas long as 12 days (288 h). Thus, specifically contemplated are methanolinductions that last about 22 h to about 12 days (288 h). For example,it is understood that methanol induction can last 163 h.

Thus an embodiment of the present invention is a method of expressing animmunotoxin in Pichia pastoris comprising a) growing the Pichia pastorisin a growth medium comprising an enzymatic digest of protein and yeastextract; b) performing methanol induction of the Pichia pastoris,wherein the methanol induction comprises a limited methanol feed of0.5-0.75 ml/min/10 L of initial volume, wherein the induction isperformed at a temperature below 17.5° C., antifoaming agent supplied upto 0.07%, and agitation is reduced to 400 RPM, and wherein the inductionstep is performed for between about 22 and 288 h.

More specifically, an embodiment of the present invention comprises amethod of expressing an immunotoxin in Pichia pastoris comprising a)growing a Pichia pastoris that expresses an immunotoxin in a growthmedium comprising about 4% glycerol, about 2% yeast extract, about 2%enzymatic digest of soy protein, about 1.34% yeast nitrogen base withammonium sulfate andwithout amino acids, and about 0.43% PTM1 solution,wherein the growth occurs at a pH of about 3.5, and wherein thedissolved oxygen concentration in the growth medium is maintained at avalue of 40% or higher; and b) performing methanol induction of thePichia pastoris, wherein the methanol induction comprises a limitedmethanol feed of 0.5-0.75 ml/min/10L of initial volume, wherein theinduction is performed at a temperature is 15° C., wherein the pH isabout 7.0, wherein antifoaming agent supplied at 0.02%, wherein theagitation reduced to 400 RPM, and wherein the induction phase is about163 h.

The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G₄S), whichselectively kills human T cells, was developed for treatment of T-cellleukemia, autoimmune diseases and tolerance induction fortransplantation (U.S. patent application Ser. No. 09/573,797,incorporated by reference). The bivalent anti-T cell immunotoxin,A-dmDT390-bisFv(G₄S), consists of the first 390 amino acid residues(DT390) of diphtheria toxin (DT) and two tandem antigen-binding domains(sFv) from the anti-CD3 antibody UCHT1. Two N-glycosylation sites in theDT390 immunotoxin have been removed by introduction of two mutations(Liu et al., 2000), resulting in a non-glycoprotein with a molecularweight of 96.5 kDa. The immunotoxin can also comprise a linker moleculeto join the antibody moiety to the toxin moiety. The linker (L) can be aGly-Ser linker. The Gly-Ser linker can be but is not limited to(Gly4Ser)n or (Gly3Ser)n. More specifically, the linker can be a(Gly4Ser)₃ linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 17), also referred toherein as (G4S), or a (Gly3Ser)₄ linker (GGGSGGGSGGGSGGGS) (SEQ ID NO:18), also referred to herein as (G3S). In a preferred embodiment theimmunotoxin comprises A-dmDT390-bisFv(G4S).

The immunotoxin is sensitive to pH levels below 6.0, as shown by thefact that low pH induces an irreversible conformational change in thetranslocation domain of the DT390 moiety. The translocation domainmediates translocation of the A chain in the DT390 from the endosomes orthe plasma membrane to the cytosol in a proton dependent manner. Thecatalytic A chain is responsible for protein synthesis inhibition byADP-ribosylation of elongation factor 2 (EF-2) in the cytosol. Thisinhibition of protein synthesis is toxic to many eukaryotic cells. ThepH sensitivity of the immunotoxin restricts the use of cation exchangechromatography and affinity chromatography based on eluting with a lowpH buffer.

The use of toxin-resistant eukaryotic cells can overcome the immunotoxintoxicity. However, selection and characterization of toxin-resistanteukaryotic cells are tedious, labor intensive and time-consuming work.Furthermore, the bivalent immunotoxin production in a EF-2 mutant CHOcell expression system was limited to 5 mg/L and could not be increasedby selection for multiple gene insertions. Due to this limitation, withthree exceptions (12, 20, 25) all recombinant immunotoxin production fortherapeutic uses has been limited to E. coli production necessitatingdenaturation and refolding from inclusion bodies (6). However, refoldingof the multi-domain structure of the bivalent immunotoxin from E. coliwas inefficient and full bioactivity was not recovered (25). Also, themulti-domain structure of the bivalent immunotoxin hinders efficientproduction in Escherichia coli. Therefore, the attempt to develop arobust Pichia pastoris production system for the bivalent inamunotoxinwas driven by the inadequacy of the existing productions systems.

Pichia pastoris is a good expression system for the bivalent anti-T cellimmunotoxin A-dmDT390-bisFv as it provides optimal protein foldingcompared to prokaryotic expression systems and provides higher yieldscompared to mammalian cell expression (CHO cells). Antibody fusionproteins require correct disulfide bridges and the endoplasmic reticulumof yeast provides an oxidizing environment like that of eukaryoticantibody producing cells. The multi-domain structure of the bivalentimmunotoxin requires a eukaryotic expression system to properly foldthis complex protein. Yet most eukaryotes are sensitive to the effectsof protein synthesis inhibition upon expression of the immunotoxin.However, a budding yeast, Pichia pastoris has a certain degree oftolerance to DT (Neville et al., 1992; Woo et al., 2002) and yielded theimmunotoxin at a level of 40 mg/L in fermentor culture. The immunotoxinwas produced by fermentation of genetically engineered Pichia pastoris(JW102, renamed from pJW#2 (Woo et al., 2002)) via the secretory route.As shown in Example 41, the present method provides a yield of 120 nag/1after a 163 h induction period and the purified yield is 90.8 mg/L (seetable 6).

After gene optimization to reduce the AT content of the DNA sequence,secreted expression levels under the AOX1 promoter of 25-30 mg/L can beobtained in bioreactors after 24-44 hours of induction. Pichia pastoriswas sensitive to the toxic effects of cytosolic expressed diphtheriatoxin A chain which ADP ribosylates elongation factor 2 (EF-2) leadingto cessation of protein synthesis. Toxicity to expression ofA-dmDT390-bisFv by the secretory route was indicated by a continuousfall in methanol consumption after induction. A mixed feed of glyceroland methanol was provided to the cells. Expression of the catalyticdomain (A chain) of DT in the cytosol is lethal to Pichia pastoris. Whencells bearing the construct A-dmDT390-BisFv (UCHT1) were induced bymethanol to express the immunotoxin, nearly 50% were killed after 24hours (Woo et al., 2002). In contrast, when the same immunotoxin wasexpressed in CHO cells that had been mutated to DT resistance, no toxiceffect was observed (Liu, et al., 2000; Thompson, et al., 2001). In thecytosol of eukaryotes, the catalytic domain of DT catalyzes ADPribosylation of elongation factor 2 (EF-2), leading to inhibition ofprotein synthesis and cell death (by protein starvation and orapoptosis, Van Ness et al., 1980; Houchins, 2000). The sensitivity ofthe eukaryotic EF-2 to ADP-ribosylation by these toxins lies in thestructure of protein. EF-2 is a single polypeptide chain of about 850amino acids and is composed of two domains. The N-terminal G domain isresponsible for binding and hydrolysis of GTP that promotes translation,and the C-terminal R (or diphthamide) domain is thought to interact withthe ribosome (Kohno et al., 1986; Perentesis et al., 1992). Thediphthamide domain (FIG. 1 a) contains a histidine residue in a regionof 22 residues that are well conserved in the EF-2 of all eukaryotes.This conserved histidine is specifically modified post-translationallyto the derivative, diphthamide, which is the unique target forADP-ribosylation by DT (Van Ness et al., 1880). In S. cerevisiae, theconserved histidine can be mutated and substitutions with some other 2amino acids yielded functional EF-2s that were resistant toADP-ribosylation (Phan et al., 1993; Kimata and Kohno 1994). However,cells with EF-2 mutated at diphthamide grew more slowly than thoseexpressing wild-type EF-2. In CHO cells, a single substitution ofarginine for glycine, which is another well conserved residue located atthe 3rd position to the C-terminal side of the diphthamide, alsoprevented the formation of diphthamide (Kohno & Uchida, 1987; Foley etal., 1992) and resulted in non-ADP-ribosylatable EF-2. This mutation hadthe same effect on EF-2 of S. cerevisiae (Kimata et al., 1993). Incontrast to the mutation at diphthamide, the Gly to Arg mutation in EF-2did not affect cell growth of CHO and S. cerevisiae (Foley et al., 1992;Kimata and Kohno 1994; Kimata et al., 1993).

In order to determine if the expression level of A-dmDT390-bisFv couldbe further increased by rendering Pichia pastoris insensitive to toxin,the EF-2 gene of Pichia pastoris has been mutated so that the Gly atposition 701 was changed to Arg, which has been shown to preventADP-ribosylation of EF-2 in other organisms. The EF-2 mutagenesisrequired cloning of the gene, introduction of the in vitro mutatedsequence with a selection marker, URA3, to the genome and PCRidentification of mutated clones. The entire EF-2 gene of Pichiapastoris has been cloned and sequenced. The coding sequence of Pichiapastoris EF-2 is 2526 nucleotides coding for 842 amino acids. The Pichiapastoris EF-2 is the same as the EF-2 of S. cerevisiae and S. pombe inlength and shares 88% and 78% of identity in amino acid sequence withthese two, respectively. In contrast to these two yeasts, Pichiapastoris has only one copy of the EF-2 gene that contains a shortintron. Before the complete sequence of EF-2 was known, differentapproaches were used to mutate Pichia pastoris to obtain DT resistantstrains. All these efforts were unsuccessful due to the lack of robustselection. Based on the EF-2 sequence obtained, a pBLURA-Δ5′ mutEF-2 wasconstructed that targets Pichia pastoris EF-2 gene and introduces amutation of Gly 701 to Arg to the gene by homologous recombination. Theconstruct contains the 3′ end 1028 nucleotides of EF-2 that has beenmutagenized in vitro to contain the amino acid substitution and theauxotrophic marker URA3. A PCR detection method was also developed forfast and accurate identification of mutant clones after uracilselection. The targeted mutation strategy with construct pBLURA-Δ5′mutEF-2 allowed mutation of the EF-2 gene of Pichia pastoris with about40% of uracil positive clones being found to contain the introducedmutations. EF-2 mutants were developed with different auxotrophicmarkers, (specifically mutEF2JC308 (ade1 arg4 h is 4), mutEF2JC303 (arg4his4) and mutEF2JC307 (his4)) and demonstrated that the Gly 701 to Argmutation in EF-2 confers resistance to the cytosolic expression of DT Achain.

When EF-2 mutants were used to express A-dmDT390-bisFv under the controlof AOX1 promoter, they did not show the advantage over the non-mutatedexpressing strain JW102 in the production of the protein in shake-flask.However, in large-scale fermentation culture under conditions adoptedfrom those optimal for JW102, the production of the mutant strainYYL#8-2 [mutEF2JC307-8(2)], increased continuously for 96 hours andreached a level 1.46-fold greater than the non-mutated JW102 strain.Cell growth and methanol consumption rates of the mutant strainexpressing A-dmDT390-bisFv were the same as that of the non-expressingwild type strain. Therefore it appeared that expression ofA-dmDT390-bisFv was not toxic to the mutant strain. The EF-2 mutantsallowed expression of A-dmDT390-bisFv under the control of theconstitutive GAP promoter (P_(GAP)). In shake-flask culture, theproduction of A-dmDT390-bisFv under P_(GAP) was about 30% higher thanthat under P_(AOX1). The increase in production under P_(GAP) may bemore significant in fermentation cultures since fermentation allowscells to grow to very high density.

In the Pichia pastoris expression system, most heterologous proteinssuch as botulinum neurotoxin fragments for vaccine use (Potter et al.,2000), hepatitis B surface antigen (Hardy et al., 2000), gelatin (Wertenet al., 1999), collagen (Nokelainen et al., 2001), and insulin (Wang etal., 2001) were successfully expressed and/or secreted by using a simpledefined medium. The cytosolic expression of the catalytic domain of DTcauses protein synthesis inhibition, leading to complete cell death inthe defined medium, but not in complex media (Liu et al., 2003). Thisfinding indicates that complex media play a role in attenuation ofprotein synthesis inhibition that is caused by ADP-ribosylation of EF-2.A very low production of the bivalent immunotoxin was observed in thedefined medium but not in a complex medium in shake flask culture.Fermentation of Pichia pastoris for expression of heterologous proteinshad been developed on the basis of a defined medium but use of complexmedia for expression of the bivalent immunotoxin in a secreted formprovides a higher level of production.

In the present large scale production of bivalent immunotoxin in Pichiapastoris, lowering the induction temperature to 15° C. substantiallyimproved the secretion of bioactive immunotoxin, and thereby compensatedfor the limitation in Pichia pastoris secretory capacity. In addition,the use of complex medium containing yeast extract further enhancedimmunotoxin secretion, apparently by attenuating the toxic effects ofthe immunotoxin on the Pichia pastoris host.

The expression level of the bivalent immunotoxin was improved by 4-foldin bioreactor culture compared to shake flask culture by optimizing thefermentation conditions in Pichia pastoris as follows: (1) use ofSoytone Peptone and yeast extract based complex medium, (2) use ofmethanol/glycerol mixed feed (4:1) to supplement the energy sourceduring methanol induction, (3) continuous feeding of PMSF and yeastextract during induction, and (4) lowering temperature to 15° C. duringmethanol induction. The lowered temperature resulted in a 2-foldincrease in secretion relative to using 23° C. during methanolinduction.

As noted above, a major problem in production of the bivalentimmunotoxin was reduction of methanol utilization during the methanolinduction phase. The reduction of methanol utilization results from areduction in the activity of the rate limiting enzyme, alcohol oxidase(AOX1). This could be secondary to protein synthesis inhibition by thebivalent immunotoxin reaching the cytosol compartment through leakagefrom the secretory compartment or by proton dependent translocation fromthe mildly acidic secretory compartment (Arata et al., 2002). The factthat methanol utilization is not affected by immunotoxin production in aPichia pastoris strain mutated to toxin resistance in the EF-2 gene (Liuet al., 2003) indicates that toxin induced ADP-ribosylation is the causeof the decreased AOX1 activity in strain JW 102. However, control ofAOX1 level is balanced by both synthesis as well as degradation, anddegradative mechanisms could be augmented in response to toxin mediatedADP-ribosylation. For reasons unknown, yeast extract increased methanolutilization, though not to wild type levels. In addition, low methanolutilization negatively affected Pichia pastoris cell growth. This wascorrected in the present method by adding another carbon source,glycerol, and continuous feeding of yeast extract during methanolinduction. These two corrections raised the methanol consumption to 80%of the non-expressing strain.

To further compensate for Pichia pastoris protein synthesis inhibitionby the expressed immunotoxin, the fermentation conditions weremanipulated for full activation of alcohol oxidase I (AOX1), the ratelimiting enzyme for methanol metabolism (Veenhuis et al., 1983). Sincethe immunotoxin gene was under the control of the same strong promoteras the AOX1 gene, the immunotoxin should be highly expressed. However,it has previously been observed in the secretion of heterologousproteins that each protein appears to have an optimal secretion level.Expression beyond the optimal level (overexpression) of secretedheterologous proteins can cause a reduction in secreted protein yieldsin mammalian, insects and yeast cells (Bannister and Wittrup, 2000;Liebman et al., 1999; Liu et al., 2003; Pendse et al., 1992). In orderto determine whether the bivalent immunotoxin was being overexpressed inPichia pastoris, the induction temperature was lowered during methanolinduction. Since most cellular activities including protein synthesisare decreased at low temperature, lowering induction temperature shoulddecrease the synthetic rate of the bivalent immunotoxin. Any resultingchange in secretion rate was judged. Bivalent immunotoxin expression wasincreased at low induction temperatures, reaching a maximum at 17.5° C.,and secretion of bioactive immunotoxin reached a maximum at 15° C., inspite of the fact that methanol consumption rate at 15° C. fell to 75%of its 23° C. value. Because continuous feeding of PMSF and yeastextract during induction effectively inhibited protease activity insupernatants, it appears unlikely that a reduction in protease activitywith lower induction temperature accounts for the nearly 2-fold increasein bivalent immunotoxin secretion seen at 15° C. The limitation inPichia pastoris secretion of bivalent immunotoxin previously describedmay actually represent an overexpression at 23° C. that is reduced at15° C. achieving a better balance of input and output within thesecretory compartment.

In short, the immunotoxin was produced in Pichia pastoris (JW102) viathe secretory route under control of the AOX1 promoter in the fermentorusing methanol as a carbon source. There were two major impediments toefficient immunotoxin production, the toxicity of the immunotoxintowards Pichia pastoris and the limited secretory capacity of Pichiapastoris for the immunotoxin. The toxicity towards Pichia pastorisresulted in a decrease in the metabolic rate of methanol consumption, acell growth rate reduction and very low productivity in a defined mediumduring methanol induction. These problems were overcome by (1) using anenzymatic digest of soy protein (e.g., Soytone peptone) and yeastextract based complex medium, (2) using methanol/glycerol mixed feed(4:1) to supplement energy source during methanol induction, and (3)continuously feeding PMSF and yeast extract during methanol induction.Lowering the induction temperature to 15° C. improved secretedimmunotoxin yield by almost 2-fold, up to 40 mg/L (at 67 hoursinduction) compared to secretion at a induction temperature of 23° C.,even though methanol consumption was reduced. In addition, with the useof the present method, the fraction of immunotoxin present asbiologically inactive oligomeric forms was decreased.

Also provided by the invention is a method of purifying anon-glycosylated immunotoxin comprising (a) loading a solutioncontaining the non-glycosylated immunotoxin onto a hydrophobicinteraction column; (b) obtaining a first non-glycosylated immunotoxincontaining eluant from the hydrophobic interaction column; (c) loadingthe non-glycosylated immunotoxin containing eluant from step (b) onto ananion exchange column; (d) obtaining a second non-glycosylatedimmunotoxin containing eluant from the anion exchange column by elutingthe non-glycosylated immunotoxin with a sodium borate solution; (e)diluting the concentration of sodium borate in the secondnon-glycosylated immunotoxin containing eluant from step (d) to about 50mM or less; (f) concentrating the diluted non-glycosylated immunotoxincontaining eluant from step (e) over an anion exchange column; and (g)obtaining a purified non-glycosylated immunotoxin from the anionexchange column. Optionally, the method further comprises washing theanion exchange column with about 25 mM sodium borate solution prior toeluting with the sodium borate solution. Preferrably thenon-glycosylated immunotoxin being purifed is expressed by the methodstaught herein.

The concentration of the sodium borate solution in step (d) of thepurification method is between about 25 mM and about 200 mM, andpreferably is between about 75 mM and about 100 mM. For example, theconcentration of sodium borate in step (e) can be about 20 mM.

A major problem encountered in the large scale purification of thebivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G₄S), from Pichiapastoris supernatants is the presence of host glycoproteins exhibitingsimilar charge, size and hydrophobicity characteristics. This problemwas overcome by employing borate anion exchange chromatography. Borateanion has an affinity for carbohydrates and imparts negative charges tothese structures. At a concentration of sodium borate between 50 and 100mM, the non-glycosylated immunotoxin did not bind to Poros 50 HQ anionexchanger resin, but glycoproteins, including aggregates related to theimmunotoxin, did bind. By using this property of the immunotoxin in thepresence of sodium borate, a 3-step purification procedure wasdeveloped: (1) Butyl 650M hydrophobic interaction chromatography, (2)Poros 50 HQ anion exchange chromatography in the presence of borate, and(3) Q anion exchange chromatography. This procedure has severaladvantages: (1) it is a relatively simple process without any dialysisor diafiltration step; (2) it exhibits good repeatability; (3) the finalyield is over 50%; and (4) the final purity is over 98%. Previously,boronic acid resins have been used to separate glycoproteins fromproteins. However, combining borate anion with conventional anionexchange resins accomplishes separation of the immunotoxin fromglycoproteins, and eliminates the need to evaluate non-standard resinswith respect to good manufacturing practice guidelines. Thus, borateanion exchange chromatography was used for separation of the immunotoxinfrom Pichia pastoris glycoproteins.

The immunotoxin is functionally active only in its monomeric form.However the supernatant harvested from the fermentation run containedmonomeric, dimeric and higher oligomeric forms of the immunotoxin aswell as Pichia pastoris proteins.

Among these Pichia pastoris proteins, a glycoprotein species ofapproximately 45 kDa was present in dimeric form (˜90 kDa). The dimericand higher oligomeric forms of the immunotoxin were relatively easy toseparate by the use of conventional hydrophobic interactionchromatography and anion exchange chromatography. However, it wasdifficult to isolate the pure monomeric immunotoxin because the 45 kDaglycoproteins were very similar to the monomeric immunotoxin in size,hydrophobicity, and isoelectric point.

Previously, immobilized phenylboronate resins have been used forseparation of glycoproteins from proteins (Myohanen et al., 1981;Bouritis et al., 1981; Williams et al., 1981; Zanette et al., 2003).These immobilized resins bind and selectively retard glycoproteinsdepending on pH, presence of sugar, type of sugar, concentration ofsugar and buffer species. Borate anion exchange chromatography is usedrather than the immobilized phenylboronate affinity chromatography forseparation of the immunotoxin from the 45 kDa glycoprotiens, because ofpoor separation capability of phenylboronate resin. Borate formscomplexes with sugar residues having vicinal cis-hydroxyl groups(Boeseken, 1949) and these complexes are reversible (Weigel, 1963).Reversible complex formation of borate with carbohydrate onglycoproteins resulted in an increased negative charge of theglycoproteins. This property allowed separation of non-glycoproteins andglycoproteins on anion exchange chromatography (Nomoto et al., 1982;Nomoto and Inoue, 1983).

In the separation of the immunotoxin from glycoproteins, borate anionexchange chromatography had different binding characteristics fromphenylboronate affinity chromatography. In phenyloboronate affinitychromatography, glycoproteins as well as the immunotoxin were boundunder the condition of low ionic strength and they were co-eluted byeither 0-100 mM sodium borate gradient or 0-50 mM sorbitol gradient,indicating that the immunotoxin physically interacts with at least oneof the bound glycoproteins, or interacts with the phenylboronate columnthrough an alternate mechanism. The fact that purified bivalentimmunotoxin also bound to the phenylboronate column indicates bindingthrough an alternate mechanism.

In previous purification methods utilizing shake flask culture, a 2-stepprocedure was employed which involved DEAE anion exchange chromatographyand Protein L affinity chromatography for purification of theimmunotoxin (Woo et al., 2002). However, the supernatants of highdensity fermentor cultures of Pichia pastoris contain materials thatseverely reduce the capacity of anion exchange resins. In addition, theProtein L affinity step required excessive column size, was expensiveand was not available under Good Manufacturing Practices (GMP)certification. Consequently, alternative procedures were developed.Hydrophobic interaction chromatography using Butyl 650M worked well as acapture step but also concentrated P. pastoris glycoproteins havingsimilar charge, size and hydrophobicity as the immunotoxin. ConcanavalinA affinity resin was promising for glycoprotein removal, but bleeding ofpotentially toxic concanavalin A from the resin resulted in unacceptablecontamination of the final product.

The anion exchange column may be but is not limited to an anion acrylic,anion agarose, anion cellulose, anion dextran or anion polystyrene. Thepreferred anion exchange columns are a Poros HQ 50 and a Q anionexchange column. By using the Poros 50 HQ borate exchange chromatographyin the present invention, substantial purification of the monomeric formof the immunotoxin was obtained, even though the immunotoxin in theeluted faction was diluted. Thus, a subsequent concentration step by Qanion exchange chromatography was used.

The hydrophobic interaction column may be but is not limited to aPhenyl-SEPHAROSE® CL-4B, Octyl Agarose, Phenyl-Sepharose 6 Fast Flow,Phenyl-Agarose, Phenyl-Sepharose 6 Fast Flow, Octyl-Sepharose 4 FastFlow, Butyl Sepharose™ 4 Fast Flow, Octyl Agarose, Phenyl-Agarose,Hydrophobic chromatography media—monoamino MAA-8, Hexyl-Agarose,Dodecyl-Agarose, Hexyl-Agarose, 4-Phenylbutylamine-Agarose,Ethyl-Agarose, Matrix, Butyl-Agarose, Propyl Agarose, Affinitychromatography media AAF-8, Octyl Agarose, Butyl-Agarose, Decyl-Agarose,Phenyl-Agarose, Methyl Matrix: Ceramic HyperD F Hydrogel Composite,Octyl Agarose, Trityl-Agarose, Q Sepharose, Ether 650, Phenyl 650, Butyl650 or Hexyl 650. The preferred hydrophobic interaction column is aButyl 650M hydrophobic interaction column.

The present borate anion exchange chromatography is useful for thepurification of other Pichia pastoris expressed proteins. Pichiapastoris is being increasingly used as an expression system fortherapeutic recombinant proteins (Cereghino et al., 2002). Many of theserecombinant proteins have their glycosylation sites removed due to theprofound differences in glycosylation patterns between Pichia pastorisand humans. These recombinant proteins are then amenable to purificationusing borate anion exchange chromatography.

It is contemplated that any buffer, flow rate, and column size may beused that would successfully effect elution of the immunotoxin in a morepure state than the immunotoxin was loaded upon the column.

An immunotoxin used in the present invention comprises a mutant toxinmoiety (e.g., DT toxin) linked to an antibody moiety (targeting moiety).Toxins that may be used include but are not limited to diphtheria toxin,ricin toxin, and pseudomonas exotoxin. The antibody moiety is preferablya single chain (sc) variable region.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, protein chemistry and immunology, which are within theskill of the art. Such techniques are explained fully in the literature,including Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nded. (Cold Spring Harbor Laboratory Press, 1989); DNA CLONING, Vol. I andII, D. N Glover ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M. J.Gait ed. (IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B. D. Hames & S.J. Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION, B. D.Hames & S. J. Higgins eds., (IRL Press, 1984); ANIMAL CELL CULTURE, R.I. Freshney ed. (IRL Press, 1986); IMMOBILIZED CELLS AND ENZYMES, K.Mosbach (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULARCLONING, Wiley (1984); the series, METHODS IN ENZYMOLOGY, AcademicPress, Inc.; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J. H. Miller andM. P. Calos eds. (Cold Spring Harbor Laboratory, 1987); METHODS INENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and Wu, ed.,respectively (Academic Press, 1987), IMMUNOCHEMICAL METHODS IN CELL ANDMOLECULAR BIOLOGY, R. J. Mayer and J. H. Walker, eds. (Academic PressLondon, Harcourt Brace U.S., 1987), PROTEIN PURIFICATION: PRINCIPLES ANDPRACTICE, 2nd ed. (Springer-Verlag, N.Y. (1987), and HANDBOOK OFEXPERIMENTAL IMMUNOLOGY, Vol. I-IV, D. M. Weir et al., (BlackwellScientific Publications, 1986); Kitts et al., Biotechniques 14:810-817(1993); Munemitsu et al., Mol. and Cell. Biol. 10:5977-5982 (1990).

The present invention utilizes a nucleic acid encoding a diphtheriatoxin-containing fusion protein, wherein the nucleic acid can beexpressed by a yeast cell. The nucleic acid capable of being expressedby yeast, comprises a modified native diphtheria-encoding sequence. Topromote expression of the nucleic acids of the present invention byyeast cells, regions of the nucleic acid rich in A and T nucleotides aremodified to permit expression of the encoded immunotoxin fusion proteinby yeast. For example, such modifications permit expression by Pichiapastoris. The modifications are designed to inhibit polyadenylationsignals and/or to decrease early termination of RNA transcription. Morespecifically, one or more AT rich regions of the nativediphtheria-encoding sequence are modified to reduce the AT content. TheAT rich regions include regions of at least 150 contiguous nucleotideshaving an AT content of at least 60% or regions of at least 90contiguous nucleotides having an AT content of at least 65%, and the ATcontent of the AT rich regions is preferably reduced to 55% or lower.The AT rich regions also include regions of at least 150 contiguousnucleotides having an AT content of at least 63% or regions of at least90 contiguous nucleotides having an AT content of at least 68%, and theAT content of the AT rich regions is reduced to 55% or lower. The nativediphtheria-encoding sequence preferably is further modified to encode adiphtheria toxin truncated at its C-terminal. Furthermore, the nativediphtheria-encoding sequence preferably is further modified to encodeone or more amino acids prior to the amino terminal glycine residue ofthe native diphtheria toxin. Furthermore, the native diphtheria-encodingsequence preferably is further modified to encode the alpha matingfactor signal peptide or a portion thereof.

The immunotoxin of the present invention may be expressed in andpurified from various organisms. These organisms include yeast such asPichia pastoris or Saccharomyces cerevisiae, bacteria such asEscherichia coli, mammalian cells such as Chinese hamster ovary cells orbaculovirus infected insect cells. There are several advantages to yeastexpression systems, which include, for example, Saccharomyces cerevisiaeand Pichia pastoris. First, evidence exists that proteins produced in ayeast secretion systems exhibit correct disulfide pairing. Second,efficient large scale production can be carried out using yeastexpression systems. The Saccharomyces cerevisiae pre-pro-alpha matingfactor leader region can be used to direct protein secretion from yeast(Brake, et al. (82)). The leader region of pre-pro-alpha mating factorcontains a signal peptide and a pro-segment which includes a recognitionsequence for a yeast protease encoded by the KEX2 gene: this enzymecleaves the precursor protein on the carboxyl side of a Lys-Argdipeptide cleavage signal sequence. The nucleic acid coding sequence canbe fused in-frame to the pre-pro-alpha mating factor leader region. Thisconstruct can be put under the control of a strong transcriptionpromoter, such as the alcohol dehydrogenase I promoter, alcohol oxidaseI promoter, a glycolytic promoter, or a promoter for the galactoseutilization pathway. The nucleic acid coding sequence is followed by atranslation termination codon which is followed by transcriptiontermination signals. Alternatively, the nucleic acid coding sequencescan be fused to a second protein coding sequence, such as Sj26 orbeta-galactosidase, used to facilitate purification of the fusionprotein by affinity chromatography. The insertion of protease cleavagesites to separate the components of the fusion protein is applicable toconstructs used for expression in yeast.

Diphtheria toxin is toxic to yeast when the toxin A chain is synthesizedwithin the cytosol compartment without a secretory signal (Parentesis etal., 1988). This toxin-catalyzed activity is specific for EF-2 andoccurs at a unique post-translational histidine residue at the position699, found in a conserved amino acid sequence in the EF-2 of alleukaryotes. A change of glycine to arginine residue at the position 701in yeast EF-2 results in resistance to DT.

In an alternative purification method, (Ala)dmDT390-bisFv(UCHT1) wasproduced in the Pichia medium at a level of 5 mg/ml whether or not themutant EF-2 gene was present. There is an extremely tight couplingbetween the presence of the alpha-mating factor signal sequence and thecompartmentalization of (Ala)dmDT390-bisFv(UCHT1) into the secretorypathway and away from the EF-2 toxin substrate in the cytosolcompartment, since one molecule of toxin in the cytosol can inactivate99% of the EF-2 in 24 hours. Producing (Ala)dmDT390-bisFv(UCHT1) inPichia utilizing the alpha-mating factor signal sequence withoutmutating the Pichia to toxin resistance provided a successful outcome.Another combination of a yeast produced toxin (ricin A chain) and signalsequence, Kar2, resulted in death of the producing cells (Simpson etal., 1999 (80)). It is possible that, at higher gene dosages ofimmunotoxin fusion protein in Pichia, mEF-2 may confer a benefit inproduction. A further advantage of yeast over mammalian cells forimmunotoxin fusion protein production is the fact that intact yeast arehighly resistant to diphtheria toxin present in the external medium tolevels as high as 3.3×10⁻⁶ M. Evidently the yeast capsule preventsretrograde transport of toxin back into the cytosol compartment asoccurs in mammalian cells and in yeast spheroplasts (Chen et al. 1985).

The invention may utilize a cell comprising a nucleic acid that encodesthe immunotoxin fusion protein. The cell can be a prokaryotic cell,including, for example, a bacterial cell. More particularly, thebacterial cell can be an E. coli cell. Alternatively, the cell can be aeukaryotic cell, including, for example, a Chinese hamster ovary (CHO)cell (including for example, the DT resistance CHO—K₁ RE 1.22 c cellline, as selected by Moebring & Moehring), myeloma cell, a Pichia cell,or an insect cell. The immunotoxin fusion protein coding sequence can beintroduced into a Chinese hamster ovary (CHO) cell line, for example,using a methotrexate resistance-encoding vector, or other cell linesusing suitable selection markers. Presence of the vector DNA intransformed cells can be confirmed by Southern blot analysis. Productionof RNA corresponding to the insert coding sequence can be confirmed byNorthern blot analysis. A number of other suitable host cell lines havebeen developed and include myeloma cell lines, fibroblast cell lines,and a variety of tumor cell lines such as melanoma cell lines.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer,and necessary information processing sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Preferred expression control sequences arepromoters derived from immunoglobulin genes, SV40, Adenovirus, BovinePapilloma Virus, etc. The vectors containing the nucleic acid segmentsof interest can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transformation is commonly utilized for prokaryotic cells,whereas calcium phosphate, DEAE dextran, or lipofectin mediatedtransfection or electroporation may be used for other cellular hosts.

The nucleic acids used in the present invention can be operativelylinked to one or more of the functional elements that direct andregulate transcription of the inserted nucleic acid and the nucleic acidcan be expressed. For example, a nucleic acid can be operatively linkedto a bacterial or phage promoter and used to direct the transcription ofthe nucleic acid in vitro.

A mutant strain of Pichia pastoris is provided. The mutant straincomprises a mutation in at least one gene encoding elongation factor 2(EF2). This mutation comprises a Gly to Arg replacement at a positiontwo residues to the carboxyl side of the modified histidine residuediphthamide. In this manner, the strain is made resistant to the toxicADP-ribosyating activity of diphtheria toxin.

A method of expressing a diphtheria toxin protein moiety is provided.Such a method of the invention comprises transfecting a mutated Pichiapastoris cell of the invention with a vector comprising a toxinprotein-encoding nucleic acid under conditions that permit expressisonof the protein-encoding nucleic acid in the cell. The conditions arethose used for Pichia pastoris cells and can be optimized for theparticular system.

The antibody moiety preferably routes by the anti-CD3 pathway or other Tcell epitope pathway. The immunotoxin can be monovalent, but bivalentantibody moieties are presently preferred since they have been found toenhance cell killing by about 15 fold. It is contemplated that anynumber of chemical coupling or recombinant DNA methods can be used togenerate an immunotoxin of the invention. Thus, reference to a fusiontoxin or a coupled toxin is not necessarily limiting. The immunotoxincan be a fusion protein produced recombinantly. The immunotoxin can bemade by chemical thioether linkage at unique sites of a recombinantlyproduced bivalent antibody (targeting moiety) and a recombinantlyproduced mutant toxin moiety. The targeting moiety of the immunotoxincan comprise the human μCH2, μCH3 and μCH4 regions and VL and VH regionsfrom murine Ig antibodies. These regions can be from the antibody UCHT1so that the antibody moiety is scUCHT1, which is a single chain CD3antibody having human μCH2, μCH3 and μCH4 regions and mouse variableregions as shown in the figures. Numerous DT mutant toxin moieties arecontemplated, including for example, DT390 and DT389, with a variety ofmutations or as the wild type toxin moiety.

The toxin moiety retains its toxic function, and membrane translocationfunction to the cytosol in full amounts. The loss in binding functionlocated in the receptor binding domain of the protein diminishessystemic toxicity by reducing binding to non-target cells. Thus, theimmunotoxin can be safely administered. The routing function normallysupplied by the toxin binding function is supplied by the targetingantibody anti-CD3. The essential routing pathway is (1) localization tocoated pits for endocytosis, (2) escape from lysosomal routing, and (3)return to the plasma membrane.

Any antibody that can route in this manner will be effective with thetoxin moiety, irrespective of the epitope to which the antibody isdirected, provided that the toxin achieves adequate proteolyticprocessing along this route. Adequate processing can be determined bythe level of cell killing.

When antibodies dissociate from their receptors due to changes inreceptor configuration induced in certain receptors as a consequence ofendosomal acidification, they enter the lysosomal pathway. This can beprevented or minimized by directing the antibody towards an ecto-domainepitope on the same receptor which is closer to the plasma membranes(Ruud, et al. (1989) Scand. J. Immunol. 29:299; Herz et al. (1990) J.Biol. Chem. 265:21355).

The mutant DT toxin moiety can be a truncated mutant, such as DT390,DT389 or DT383, or other truncated mutants, with and without pointmutations or substitutions, as well as a full length toxin with pointmutations, such as DTM1, or CRM9 (cloned in C. ulcerans), scUCHT1 fusionproteins with DTM1 and DT483, DT390 and DT389, and have been cloned andexpressed in E. coli. The antibody moiety can be scUCHT1 or otheranti-CD3 or anti-T cell antibody having the routing and othercharacteristics described in detail herein. Thus, one example of animmunotoxin for use in the present methods comprises the fusion proteinimmunotoxin UCHT1 (or a fragment thereof)-DT390.

There is a consensus sequence for glycosylation (NXS/T (SEQ ID NO:19))that may be removed or inserted to control glycosylation. Glycosylationoccurs in all eukaryotes, e.g. Pichia pastoris.

There are numerous variants of the immunotoxins. Protein variants andderivatives are well understood to those of skill in the art and caninvolve amino acid sequence modifications. For example, amino acidsequence modifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Immunogenic fusion protein derivatives, such as those described in theexamples, are made by fusing a polypeptide sufficiently large to conferimmunogenicity to the target sequence by cross-linking in vitro or byrecombinant cell culture transformed with DNA encoding the fusion.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein molecule.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingare referred to as conservative substitutions.

Amino Acid Abbreviations

Amino Acid Abbreviations Alanine Ala, A Allosoleucine AIle Arginine Arg,R Asparagine Asn, N aspartic acid Asp, D Cysteine Cys, C glutamic acidGlu, E Glutamine Gln, K Glycine Gly, G Histidine His, H Isolelucine Ile,I Leucine Leu, L Lysine Lys, K Phenylalanine Phe, F Proline Pro, PPyroglutamic acid PGlu Serine Ser, S Threonine Thr, T Tyrosine Tyr, YTryptophan Trp, W Valine Val, V

Amino Acid Substitutions

Original Residue Exemplary Conservative Substitutions, others are knownin the art.

Ala ser Arg lys, gln Asn gln, his Asp glu Cys ser Gln asn, lys Glu aspGly pro His asn, gln Ile leu, val Leu ile, val Lys arg, gln; Met leu,ile Phe met, leu, tyr Ser thr Thr ser Trp tyr Tyr trp, phe Val ile, leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in theamino acid substitution table, i.e., selecting residues that differ moresignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce the greatestchanges in the protein properties will be those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or praline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, areaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T.E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]),acetylation of the N-terminal amine and, in some instances, amidation ofthe C-terminal carboxyl.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases.

It is also preferred that the transcribed units contain other standardsequences alone or in combination with the above sequences improveexpression from, or stability of, the construct.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Transformation with a Mutagenizing Oligonucleotide

The oligomer of 56 nucleotides (see List of Primers) contains two pointmutations to change amino acid 701 from glycine to arginine. Amutagenizing oligo (56 mer, 100 ug) was co-transformed into the GS200(Mut+, His−, Arg−) strain with an ARG4 DNA fragment. The ARG4 gene withpromoter was taken from plasmid PMY30 (supplied by Prof. Jim Cregg, KeckGraduate Institute of Applied Life Sciences, Claremont, Calif. 91711) bySph I and EcoR V. Approximately 1000 transformants were obtained. Toscreen for mutated clones having the correct mutations, diagnostic PCRwith primers mdb1EF-2 and 2253EF-2C was employed. The mutation-detectingprimer (mdb1EF-2) can distinguish a difference in DNA sequence betweenthe normal gene and the mutated gene at amino acid 701. For the normalgene, a PCR product could not be produced because 2 nucleotides at the3′ end were not matched with the DNA sequence of the normal gene,preventing extension by the Taq polymerase. For the mutated gene, theprimer could anneal perfectly, so Taq polymerase could produce a PCRproduct. More than 1000 colonies were screened by this PCR method but nomutated colony was identified. (In the above PCR assay, amino acid 701mutated EF-2 from S. cerevisiae served as a positive control. Thismutated gene had been made previously with the intent of introducing itinto Pichia pastoris. However, Pichia pastoris thus transformed had avery slow growth rate and produced the protein of interest at lowlevels.)

Transformation was performed with a partial DNA fragment containing theconserved region of the EF-2 gene and a mutation on amino acid 701. Thepartial sequence of Pichia pastoris EF-2 (positions 1717 to 2289, FIG. 3(a) (b) (c) (d)) was mutated in vitro to change the amino acid 701 fromglycine to arginine (FIG. 1 b) and then co-transformed into the GS200strain with the ARG4 gene fragment. More than 2000 Arg4 positivetransformants were obtained and screened them for the EF-2 mutation bydiagnostic PCR with primers mdb2EF-2 and 2253EF-2C. The mutation was notobserved.

List of Primers

Primers derived from S. cerevisiae EF-2:

5′ primer: (SEQ ID NO: 20) TTG GTT ATT GAC CAA ACT AAG GCT GTCCAA 3′primer: (SEQ ID NO: 21) ACC TCT CTT CTT GTT TAA GAC GGA GTA GATPrimers used in cloning and mutation of Pichia pastoris EF-2

dT₂₂-Not: (SEQ ID NO: 22) 5′-CTT GCT TTT GCG GCC GCT TTT TTT TTTTTT TTT TTT TTT EF-2C₂: (SEQ ID NO: 23)5′-G ATA AGA ATG CGG CCG CCA TTT CTT GGT CTT TGG GTT GAA G EF-2C₁:(SEQ ID NO: 24) 5′-GAT AAG AAT GCG GCC GCC AAC TTA GTTGTT GAC CAG TCT AAG 5′EF-2: (SEQ ID NO: 25)5′-ATA GCT AGC ACT TTG AAG TTC TTA ATT TTG TTC CTC 3′EF-2C:(SEQ ID NO: 26) 5′-ATA AGA ATG CGG CCG CAA GTT AAT GAAACA TTA AGC TTA CAA C wEF-2: (SEQ ID NO: 27)5′-G AAT GAC TTG TCC TCC ACC mEF-2: (SEQ ID NO: 28)5′- G AAT GAC TTG TCC TCC GCG G EF-1426: (SEQ ID NO: 29)5′-CAA CTA GCT AGC GCT CAC AAC ATG AAG GTC ATG AAA TTC BF-1318:(SEQ ID NO: 30) 5′-AGA ACC GTC GAG CCT ATT GAC GATMutagenizing oligo:

(SEQ ID NO: 31) 5′-CC CTG CAC GCC GAT GCT ATC CAC AGAAGA GGA GGA CAA GTC ATT CCA ACC ATG AAG mdb1EF-2: (SEQ ID NO: 32)5′-GCC GAT GCT ATC CAC AGA AGA mbb2EF-2: (SEQ ID NO: 33)GCC GAT GCT ATC CAC CGC CGC 2253EF-2C: (SEQ ID NO: 34)TCT CTT CTT GTT CAA AAC AGA GTA GAT ACC

Example 2 Spheroplast Transformation with the Partial Fragment ofMutated EF2 and ARG4 Fragment

In the methods of Example 1, there was no selection step against wildtype DT. A double transformation was thus employed. First, the mutatedEF-2 fragment was transformed into the GS200 strain by electroporation.Then, electroporated cells were cultivated overnight to allow theexpression of mutated EF-2 inside cell. Cultivated cells were used formaking spheroplasts. The resulting spheroplasts were treated with wildtype DT (200 μg/ml) and ARG4 fragment (10 μg) for 1 hour and transformedby CaCl₂ and PEG. Only a few transformants of normal colony size wereobtained and there was no mutated strain. In addition, there were 100 ormore micro-colonies obtained. 100 of these were screened but the mutatedstrain was not detected.

Example 3 Cloning and Sequencing of EF-2 Gene from Pichia pastoris

Prior to the cloning of the full sequence of the Pichia pastoris EF-2gene, a partial sequence had been obtained. Initially, the conserved Rdomain of Pichia pastoris EF-2 was amplified from the genomic DNA usingtwo primers derived from the same region of S. cerevisiae EF-2(Perentesis et al., 1992). The 5′ primer contained the sequence fromposition 1933 to 1962 of S. cerevisiae EF-2, whereas the 3′ primer wascomplementary to the region of 2227 to 2256. The sequence of 324nucleotides was then extended towards the 5′ end to position 284 and the3′ end to position 2289 in the coding region of Pichia pastoris EF-2gene. The extended sequence was later found to contain several mistakes.To clone the entire Pichia pastoris EF-2 gene, two species of cDNA werefirst synthesized separately from EF-2 mRNA with two different primers.Primer dT22-Not contains a run of 22 T residues complementary to the 3′polyA tail of the mRNA and the recognition sequence for restrictionenzyme Not 1. Primer EF-2C2 has 25 nucleotides complementary tonucleotide positions 747 to 771 (FIG. 3 (a) (b) (c) (d)) of the Pichiapastoris EF-2 coding sequence. After cDNA synthesis, a homopolymerictrack of A residues was added to the 3′ end of the cDNA extended fromprimer C2 by homopolymeric tailing (Sambrook et al., 1989). The 5′ endsequence of EF-2 was amplified by PCR from the modified cDNA with EF-2C2and dT22-Not primers, whereas the 3′ end sequence was from the cDNAsynthesized from primers dT22-Not and EF-2C1, which contains 27nucleotides corresponding to the positions 1927 to 1953. The PCRproducts representing the 5′ end and 3′ end sequences of Pichia pastorisEF-2 were then separately cloned to pCR2.1-TOPO vector (Invitrogen).

Five independent clones containing 5′ sequence of EF-2 Pichia pastoriswere selected for sequencing. They were first sequenced with M13 reverseand M13 forward primers located in the vector close to the up and downstreams of the insert respectively, and then with an internal primecomplementary to the positions 349 to 384 of EF-2 coding sequence. Amongthe 5 clones, three had identical sequences, one had two differentnucleotides at two different internal locations, and the other one hadanother different internal nucleotides at a different location. Thesedifferent nucleotides were likely produced by the cloning proceduressince none of these different nucleotides were present in the clonederived from genomic DNA. At the 5′ end, all five clones also had 57 to69 nucleotides of the same sequence before the first ATG codon. Thelargest open reading frame (ORF) of the 5′ end sequence starting fromthe first AUG is 747 nucleotides and the deduced amino acid sequence(249 aa) shares 90% identity with the first 249 aa at the N-terminus ofS. cerevisiae EF-2. All four clones containing the 3′ end sequence ofthe EF-2 sequence had the same sequence of 675 nucleotides followed by ahomopolymeric A track. The largest ORF is 603 nucleotides ended at stopcodon TAA, which is 72 nucleotides up stream of the poly-A track. Thededuced amino acid sequence (201 aa) shares 85% identity with the last201 aa at the C-terminus of S. cerevisiae EF-2. Having obtained both the5′ and 3′ end sequences of Pichia pastoris EF-2, two primers weredesigned to amplify the entire the EF-2 gene from the genomic DNA ofPichia pastoris. Primer 5′EF-2 is derived from the 5′ non-coding regionand contains the sequence from positions 28 to 54 relative to the ATGinitiation codon. Primer 3′EF-2C contains 27 nucleotides complementaryto positions 2523 to 2549 at the 3′ end. After PCR amplification withPfu polymerase (Stratagene), the PCR products of EF-2 gene were treatedwith Taq polymerase to have the 3′A-overhangings added (Instructionmanual for original TA cloning kit, Invitogen) and then inserted intothe TA cloning vector pCR2.1-TOPO. Ten clones were picked, and therestriction enzyme analysis of plasmid DNA isolated from these clonesindicated that they all had the same insert. DNA sequencing wasperformed first with M13 reverse and M13 forward primers and thenadvanced step by step towards the opposite ends with primers derivedfrom the sequences obtained from the previous steps. Eight clones werecompletely sequenced, and found to be identical. The 3′ end sequenceobtained from the genomic DNA is identical to that from the mRNA.However, compared to the 5′ sequence of mRNA, the sequence from thegenomic DNA has an insertion of 77 nucleotides in the codon immediatelynext to the initiation site of the EF-2 ORF (FIG. 2). The insertion hasthe sequence GTATGT CACTAAC . . . TAG (SEQ ID NO:35), a conservedpattern of short introns in S. cerevisiae (Davis et al., 200; Rymond &Rosbash, 1992). Although introns are common in S. cerevisiae, they arerarely present in Pichia pastoris (Gregg, personal communication). Thecoding sequence of Pichia pastoris EF-2 is present in FIG. 3 (a) (b) (c)(d). It contains 2526 nucleotides and code for 842 amino acids. ThePichia pastoris EF-2 is the same as the EF-2 of S. cerevisiae andSchizosaccharomyces pombe in length and shares 88% of identity in aminoacid sequence with S. cerevisiae (Perentesis et al., 1992) and 78% withS. pombe (Mita et al., 1997). Both S. cerevisiae and S. pombe have twofunctional EF-2 genes (EFT1 and EFT2) per haploid genome. These twocopies of the EF-2 genes encode the same amino acid sequence, but have afew different nucleotides (4 in S. cerevisiae and 13 in S. pombe) intheir coding regions and dissimilar flanking sequences. However, thesequencing data of independent clones derived from mRNA and genomic DNAshowed that all of the different clones had the same 5′ and 3′ endflanking sequences and an identical coding sequence. This plus theevidence of Southern blotting of restriction enzyme digested genomic DNAshows that Pichia pastoris has only one copy of the EF-2 gene.

Example 4 Construction of Mutating Plasmid pBLURA-Δ5′mutEF-2

To create DT resistant strains of Pichia pastoris, the EF-2 gene wasmutated so that the Gly at position 711 was changed to an Arg. Thestrategy employed to introduce the mutation into the genome is based onthat described by Shortle et al. (1984) and is shown in FIG. 4. In thismethod, a truncated form (at only one end) of the targeted gene was usedto introduce a mutation to the gene in the genome by homologousrecombination. Integration of the truncated gene fragment bearing amutation will lead to a situation that the genome contains one intactcopy of the gene with the mutation and one truncated copy. Because thetargeted site is located close to the 3′ end, the 5′ truncated EF-2(Δ5′EF-2) was used as the mutating sequence. Δ5′EF-2 contained 1127nucleotides from the 3′ end of EF-2 starting from position 1432 to 2549(FIG. 3) and was generated by PCR with Pfu polymerase. After cloninginto the pCR2.1-TOPO vector, Δ5′EF-2 was mutagenized in vitro byoligonucleotide-directed mutagenesis. The mutagenized Δ5′EF-2 (Δ5′mutEF-2) was then released from pCR2.1-TOPO by restriction enzymes Nhe1and Not 1 that cut at the 5′ and 3′ ends respectively, and then clonedinto the vector pBLURA-SX (provided by Professor Cregg and described inGeoffrey et al. (2001)) that had been digested by Nhe1 and Not 1. Thevector contains the auxotrophic marker URA3. Plasmid DNA pBLURA-Δ5′mutEF-2 purified from bacterial was linearized before beingelectroporated into the strains of Pichia pastoris. The plasmid DNAcontains a unique Aat II site located in the EF-2 sequence, about 220nucleotides before the mutation site. Cleavage at this site will targetthe plasmid integration to the EF-2 locus and favors the event of themutagenized sequence being transferred to the intact copy of EF-2. Threeuracil auxotrophic strains of Pichia pastoris were transformed with theplasmid DNA. They are JC308 (ade1 arg4 his4 ura3), JC303 (arg4 his4ura3) and JC307 (his4 ura3), and were all provided by Professor Creggand described in Geoffrey et al. (2001). JC308 was transformed firstfollowed by 10303 and JC307.

Example 5 Identification of Clones Containing Mutated EF-2

After electroporation with the linearized pBLURA-Δ5′ mutEF-2 DNA, Cellswere spread onto plates containing synthetic complete medium for yeastminus uracil (K.D Medical, Maryland). Ura+ clones were then analyzed by“Colony PCR” for the presence the correct mutations in the intact copyof EF-2. In this method, yeast cells from colonies were picked by toothpickers and resuspended in 20 ul of PCR mix. DNA released from the cellslysed by the first PCR step (94° C. for 5 minutes) served as thetemplate for PCR amplification. Five primers were used in the PCRdetection procedures: primers 5′EF-2 and 3′EF-2C were describedpreviously in section 4; EF-2 (1318) has the EF-2 sequence from position1318 to 1341; primer wEF-2 is complementary to the positions 2100 to2119, whereas primer mEF-2 has the sequence complementary the samepositions but specific to the mutations. The designed nucleotidemutations shown in FIG. 1 b created a new Sac II restriction enzyme sitethat was used to confirm the correct mutations in the genome.

Primers 5′EF-2 and mEF-2 were first used to detect the mutations in theUra+ transformants. FIG. 6 a shows that 9 (clones 1, 2, 3, 12, 13, 14,33, 40 and 41) of the 12 selected Ura+ clones of JC308 are mEF-2 primerpositive, they had a PCR product of the expected size (about 2.2 kbp),whereas clones 25, 38 and 47 were negative. As shown in FIG. 6 b, whenthe same clones were analyzed for the presence of wild type sequencewith primers EF-2 (1318) and wEF-2, all three mEF-2− clones were wEF-2primer positive. A 0.8 kbp PCR fragment was produced. All mEF-2+ cloneswere wEF-2− except for clones 33 and 41 that were also wEF-2+. Finallywhen primers EF-2 (1318) and 3′EF-2C were used, all of the selectedclones yielded a PCR product of about 1.2 kbp as expected (FIG. 6 c).The PCR products from the clones that were mEF-2+ and wEF-2− werecompletely digested by Sac II, whereas those of the clones 25, 38 and 47that were mEF-2. but wEF-2+ were not cut by the enzyme. In agreementwith being both mEF-2+ and wEF-2+, clones 33 and 41 produced both Sac IIclearable and non-clearable PCR produces. To investigate why clones 33and 41 had both mutated and wild type EF-2, these clones were streakedon new selection plates and let the cells grow to form colonies. Tenwell-isolated colonies were picked from each and performed the PCR withprimer EF-2 (1318) plus primer 3′EF-2C and the Sac II digestion steps.None of the colonies had the same mixed PCR products as the originals.PCR products of 4 colonies from clone 33, 7 from 41 were completelydigested by Sac II, whereas those of other colonies from clones 33 and41 were not cut at all. This experiment shows that clones 33 and 44 wereeach originally formed by two different cells, one had an intact EF-2with the mutations, and the other had an intact wild type EF-2. Thisexperiment was then repeated and checked some of the clones that hadonly the Sac II clearable EF-2 (clones 1, 2, 3, 12, 13, 14, and 40) andconfirmed that they only contained the mutated intact EP-2. After thesuccess in obtaining EF-2 mutant clones of JC308, the same selectionprocedure was used to identify EF-2 mutant clones of JC303 and JC307.Among the Ura+ positive clones picked for analysis, 35% of themcontained only the mutated intact EF-2. This high frequency of completemutation may be due to the fact that Pichia pastoris only has one copyof EF-2 per haploid genome. As shown for CHO cells and S. cerevisiae,the Arg substitution for Gly711 of EF-2 in Pichia pastoris did notaffect cell growth at normal conditions.

Example 6 Expression of DTA Chain in the EF-2 Mutants

To test whether the obtained EF-2 mutants are resistant to DTexpression, mutEF2JC307-8, an EF-2 mutant clone (clone 8) of JC307, wastransformed with the plasmid DNA of pPIC3-DtA. The construction ofpPIC3-DtA was previously described (Woo et al., 2002). Briefly, the DT Achain gene with BamH I at its 5′ end and Not I at 3′ was amplified byPCR, inserted into Pichia pastoris expression vector pPIC 3 (Invitrogen)and digested with these two enzymes. Integration of pPIC3-DtA allowscytosolic expression of DT chain upon methanol induction. This plasmidDNA had previously been used to transform the GS200 strain of Pichiapastoris (Invitrogen) and two of the resulting clones (C3 and C4) wereused in the study on tolerance of Pichia pastoris to DT (Woo at al.,2002). C3 had been characterized as a non-DT A expressing clone, whereasC4 is a DT A expressing clone. After the transformation with pPIC3-DtA,six mutEF2JC307-8, (mutEF2JC307-8-DtA(1) to (6), clones were randomlypicked for analysis of their cytosolic expression of DT A chain andtheir viability after methanol induction. Cells from single colonies ofmutEF2JC307-8-DtA(1) to (6), C3 and C4 were grown in 2 ml YPD (Yeastextracts-Peptone-Dextrose) medium at 30° C. overnight before beingpelleted down by centrifugation. Cells from each culture wereresuspended in YP medium to a density at OD600 nm±0.5. Cell suspensions(2 ml) were induced by adding methanol to 1% and incubated at 30° C.with vigorous shaking. After methanol induction for 24 hours, cells from100 μl of each culture were pelleted down and washed with PBS buffer.After this, cells were resuspended in PBS and mixed with protein samplebuffer. Finally, the samples were subjected to two cycles of boiling andfreezing on dry ice before being analyzed by SDS-PAGE and Westernblotting with a DT specific antibody. The cultures ofmutEF2JC307-8-DtA(3) and (5), C3 and C4 were also used for viabilityassay. This was performed by diluting each culture 104 to 107 fold withPBS buffer, plating 100 μl of aliquot on YPD plate and then counting thecolonies appearing on the plates after 3 days incubation at 30° C. Theresult of SDS-PAGE and Western blotting showed that except formutEF2JC307-8-DtA(5), all mutEF2JC307˜8-DtA clones expressed DT A chain(FIG. 7 a). The expression of mutEF2JC307-8-DtA(3) was estimated roughlyat 20 μg/ml cell culture. As expected, C3 did not express DT A. AlthoughC4 did express DT A, the protein band was barely visible (FIG. 7 b).Before methanol induction, the number of the colony forming units (CFU)per ml of cells was about the same for mutEF2JC307-8-DtA(3) and (5), C3and C4. After 24 hours methanol induction, the CFU number ofmutEF2JC307-8-DtA(3) and (5) and C3 all increased about 103 fold,whereas the CFU number of C4 decreased about 102 (FIG. 8). This resultdemonstrated that the expression of DT A chain in the cytosol of cellsbearing the mutated EF-2 was not toxic to the cells.

Example 7 Small-Scale Expression in Shake-Flask Culture

MutEF2JC307-8 was first used to express the bivalent immunotoxin Sincethis EF-2 mutant is auxotrophic for histidine, it was transformed withplasmid pPIC9K containing the final version of the modified gene for thebivalent immunotoxin: A-din DT390-bisFv described in Woo et al. (2002).Bivalent refers to two repeats of the sFv antibody fragment. Theprotocols used for transformation, selection for transformants, andprotein expression and analysis were described previously (Woo et al.,2002, which is incorporated herein by reference in its entirety for themethods taught therein). After transformation, 12 colonies were randomlypicked and analyzed for protein expression. SDS-PAGE analysis revealedthat all of the selected clones expressed the bivalent immunotoxin,although some clones, such as clone number 2 [mutEF2JC307-8(2)],expressed at slightly higher levels than others. When they were culturedand expressed under the same conditions and at the same time,mutEF2JC307-8 (2) expressed the bivalent immunotoxin at the same levelas pJHW#2, a clone of GS 115 (bearing the wild type EF-2) that had beentransformed with pPIC9K-A-din DT390-bisFv. The expression levels ofmutEF2JC307-8(2) and pJHW#2 were about 5 to 10 μg/ml of culturesupernatant in shake-tube culture. The fact that mutEF2JC307-8(2) didnot yield a higher level of expression demonstrated that other factorsin addition to EF-2 ADP ribosylation also limit production of thebivalent immunotoxin.

In a second attempt to express the bivalent immunotoxin in mutatedPichia pastoris, two copies of A-dmDT390-bisFv gene were introduced intomutEF2JC303-5, an EF-2 mutant clone (clone 5) of JC303, which isauxotrophic for histidine and arginine. To build an expression vectorwith ARG4 selection marker, The A-dmDT390-bisFv gene (see FIG. 20) wascloned into the expression vector pBLARG-SX3 provided by Professor Creggand described in Geoffrey et at (2001). This was done by inserting thefinal version of A-dmDT390-bisFv gene plus the α-factor signal sequencereleased from pPICZα (Woo et al., 2002) by Hind III and Not I digestioninto pBLARG-SX3 that had been cut with these two restriction enzymes.The resulting construct, pBLARG-A-dmDT390-bisFv (FIG. 9 a), togetherwith pPIC9K-A-dmDT390-bisFv were electroporated at the same time intomutEF2JC303(5). Transformants expressing these two marker genes wereselected on plates containing synthetic complete medium minus arginineand histidine (K.D Medical, Maryland). Eighteen colonies were pickedfrom the selection plate and analyzed for their expression of theimmunotoxin protein. SDS-PAGE showed that they all secreted roughly thesame amount of intact immunotoxin protein into induction media. Thisamount was similar to that secreted from single copy clones:mutEF2JC307-8(2) and JHW#2. As shown in FIG. 10 a, three of the selectedclones (clones 3, 6, 8) also expressed a smaller, but much more abundantprotein that reacted with an anti-DT antibody and had the same size asthe monovalent immunotoxin (Liu et al., 2000). The smaller protein ismore stable than the intact protein regardless as to whether thisprotein was produced from a truncated copy of A-dmDT390-bisFv gene orthe proteolytically cleaved product of the intact protein. The figurealso shows that there were many other smaller proteins in the culturesupernatant that reacted with the anti-DT antibody; they were mostlikely the proteolytic cleaved products of the intact protein. Thesmallest and also the most abundant one was characterized as the A chainof DT, which is very stable (Collier 1975) and can account for the finalproduct of proteolytic degradation of the intact protein. Thedegradation also took place inside the cell (FIG. 10 b). Because the Achain is about ¼ of the size of the intact protein, the amount of the Achain shown on the Western blot indicates that the actual expressionlevel was probably several times higher than the level of intact proteinpresent in the induction medium. A majority of the protein synthesizedwas probably degraded either before or after secretion out into themedium. Although the double copy clones accumulated the same amount ofintact protein in the medium as the single copy clones, the double copyclones produced a larger amount of degraded products, indicating thatmore gene products had been synthesized. Different measures to controlthe protein degradation have been employed but the production of theintact protein has not been increased. Thus protein degradation eitherwithin or outside the cell is a limiting factor to increase theproduction of the bivalent immunotoxin.

Example 8 Alternative Method for Large-Scale Expression in FermentationCulture Using PMSF

For large scale cultures, the BioFlo 4500 fermentor (New BrunswickScientific Company), which was installed with a methanol sensor (RavenBiotechnology Company) for precise control of methanol concentration incultures, was used. The initial fermentation medium (10 L) contained 1%yeast extract, 2% peptone or 2% soytone, 4% glycerol, 1% casamino acids,1.34% yeast nitrogen base with ammonium sulfate andwithout amino acids,0.43% PTM1 salt solution and 0.01% antifoam 289 (Sigma Co.) or a mixtureof antifoam 204, 0.01% and Stuktol 0.01%. Depending on cultureconditions, 75% (v/v) glycerol solution having 1.8% PTM1 salt solutionwas used for obtaining a desired cell density before methanol inductionand/or supplementing an additional carbon source or energy source formethanol induction. 100% methanol solution for induction containing 20mM PMSF and/or 1.2% PTM1 salt solution was used. Alternatively,induction was performed with a continuous feed of 4:1 methanol/glycerolcontaining 73 mM PMSF, and PMSF was added to 1 mM final concentrationjust prior to induction. In order to prepare a seed culture for thefermentor, 50 ml of YPD (1% yeast extract, 2% peptone and 2% glucose)was innoculated with 1 ml of a frozen stock of YYL #8-2 and thencultivated for 2 days at 30° C. with vigorous shaking. The 30 ml fromthe 50 ml culture was used as the first seed culture for inoculatingapproximately 600 ml of the second seed culture. The DO level in thefermentor was maintained at more than 25% for the whole fermentationrun. The pH in the fermentor was kept at 3.5 for growth phase and 7.0for methanol induction phase. The temperature was set at 28° C. forgrowth and 15-25° C. for methanol induction. Casamino acids solution(20%) was fed continuously at 20 ml/h during methanol induction or atthe maximum speed of a pump for feeding for the first 2 hours ofmethanol induction. At the temperature of 23° C. for methanol induction,the expression level of the bivalent immunotoxin was the highest among 4different runs. However, its expression level was similar to that of thecurrent expression strain, pJHW #2. Table 1 summarizes results of 5fermentation runs.

TABLE 1 Results of Fermentation Runs Run 1 Run 2 Run 3 Run 4 Run 5 (#27)(#28) (#29) (#36) (#41) glycerol-fed   5.5   4   0   7.5   6 batch time(hour) cell density  19.44  18.60  11.93  20.02  21.16 at the start ofmethanol induction (%) final conc. of   2¹   2¹   2¹   7²   2¹ PMSF (mM)casamino  100³  100³  100³  100⁴  138⁵ acid (g) temperature  25  20  15 23  23 for methanol induction (C) methanol 3093 2776 2474 2538 3000consumption (g) glycerol  475   0   0   0   0 feeding for methanolinduction (g) methanol  43  44  70  44  94 induction time (hour) finalvolume  13.3  12.3  11.9  11.4  13.4 of the supernatant (L) expression 10  15  10  15 NM⁶ level (mg/L) at 22 hours of induction expression NMNM NM NM  27.5 level (mg/L) at 42 hours of induction expression NM NM NMNM  30.0 level (mg/L) at 66 hours of induction Expression   3.3  18.3 26.6  27.5  32.5 level (mg/L) at harvest Total amount  43.9  225.1 316.5  313.5  435.5 of the bivalent immunotoxin (mg) ¹50 ml of PMSFsolution (3.484 g per 50 ml of methanol) was fed on the based ofmethanol concentration in the culture for the beginning of methanolinduction. After the finish of feeding of PMSF solution, methanolsolution containing 12 ml of PTM1 salt solution per 1 liter of methanolwas replaced. ²15 ml of PMSF solution (1.742 g per 15 ml of methanol)was injected at the beginning of methanol induction. On the basis ofmethanol concentration, methanol solution (20 mM PMSF and 12 ml of PTM1salt solution/liter of methanol) was fed. ³10% casamino acids solutionwas fed at the maximum speed of a pump at the start of methanolinduction. ⁴20% casamino acids solution was continuously fed at 20ml/hour of pump speed. ⁵15% casamino acids solution was continuously fedat 20 ml/hour of pump speed. ⁶not measured

Under these conditions, maximum production of the wild-type expressionstrain, pJHW #2, is 27.5 mg/L with the total amount of 286.0 mg of thebivalent immunotoxin in 42 hrs of methanol induction. This level couldnot be increased beyond 42 hrs of induction. However, under conditionsadopted from those for pJHW #2, production the EF-2 mutant strain YYL8-2continued to increase up to 94 hrs after methanol induction in spite thefact that the initial 10 L of culture medium was gradually diluted to13.4L with methanol and 10% casamino acids solution (see run 5). Thetotal amount of the bivalent immunotoxin of run 5 was 435.5 mg (32mg/L). This is 1.46-fold greater that the maximum production of pJHW #2.The difference in the production of the bivalent immunotoxin betweenthese two strains is reflected by the methanol consumption rates asshown in FIG. 11.

Example 9 Previous Method of Purification of the Bivalent Immunotoxin

The Pichia pastoris supernatant contains materials that compete withA-dmDT390-bisFv in binding to anion exchange resins. In addition, thetoxin moiety can not be exposed to pH less than 6.5 without undergoingunfolding of hydrophobic residues. Therefore a hydrophobic interactionchromatographic step using Butyl-650M (TosoHaas) was employed. Thisresin preferentially binds monomeric A-dmDT390-bisFv over the dimericform, a species having greatly diminished biologic activity. The capturestep also concentrates a Pichia pastoris glycoprotein that appears as adiffuse band of ˜40 kD on SDS gels but has the same mobility asA-dmDT390-bisFv under size exclusion chromatography. This material iseliminated by preferentially binding to Con A Sepharose (Pharmacia). ASuperdex (Pharmacia) size exclusion step eliminates any A-dmDT390-bisFvdimmer not previously screened during the capture step. The overallyield is 45% when the fermentation conditions achieve an A-dinDT390-bisFv monomer content of 85%. The procedure for purification ofA-dmDT390-bisFv is presented below:

1. Butyl-650M hydrophobic interaction chromatography

-   -   Bed volume: 600 ml (in 10 cm diameter column)    -   Flow rate: 50-70 cm/hour    -   sample preparation: solid sodium sulfate and 1 M Tris buffer (pH        8.0) were added to the final concentration of 0.5 M and 20 mM,        respectively.    -   sample volume: typically 10 L    -   binding buffer: 500 mM Na2SO4, 1 mM EDTA, 20 mM Tris buffer (pH        8.0)    -   elution buffer: 5% glycerol, 1 mM EDTA, 20 mM Tris buffer (pH        8.0)    -   procedure:        -   equilibrate the column with binding buffer        -   applied the sample onto the column        -   washed with 5 bed volume of binding buffer        -   eluted A-dmDT390-BisFv with 6 bed volume of elution buffer        -   regenerated the column by manufacturer's protocol    -   volume of eluted fractions: 3600 ml

2. Diafiltration

-   -   membrane: Amicon spiral-wound membrane (30 Kd)    -   model S3Y30 (0.23 m2)    -   sample: eluted fractions from capturing step    -   diafiltration buffer: 5% glycerol, 1 mM EDTA, 20 mM Tris buffer        (pH 8.0)    -   buffer volume used for diafiltration: 6 volume of the sample    -   pressure: 7 psi    -   final volume: around 2 L        3. Poros 50 HQ ion exchange chromatography    -   Bed volume: 40 ml (in 2.6 cm diameter column)    -   Flow rate: 1 ml/min    -   sample: diafiltrated sample (typically 2 L)    -   binding buffer: 5% glycerol, 20 mM Tris buffer (pH 8.0)    -   elution: 0˜500 mM NaCl gradient (10 bed volume) in binding        buffer    -   procedure:        -   equilibrate the column with binding buffer        -   applied the sample onto the column        -   washed with 3 bed volume of binding buffer and started to            collect 20 ml of each fraction        -   eluted A-dmDT390-BisFv with 10 bed volume of 0˜500 mM NaCl            gradient        -   regenerated the column by manufacturer's protocol    -   fraction size: 20 ml        4. Con A affinity chromatography    -   a sample: 90˜120 ml of the eluted fractions having        A-dmDT390-BisFv from Poros TEX    -   bed volume: 60 ml resin packed in 2.5 cm×20 cm column    -   binding buffer: 5% glycerol, 20 mM Tris buffer (pH 8.0)    -   flow rate: by gravity    -   procedure        -   equilibrated the column with binding buffer        -   applied the sample to the column and started to collect 10            ml of each fraction        -   added 0.5 M EDTA to each fraction at the final conc. of 1 mM        -   washed the column with 5 bed volume of binding buffer        -   regenerated the resin by manufacturer's protocol            5. Superdex 200 prep grade Gel filtration    -   sample: 50 ml pooled fraction containing A-dmDT390-BisFv from        Con A affinity step    -   sample preparation: 5 M NaCl was added to the final conc. of 200        mM    -   bed volume: 970 ml of Superdex 200 resin in 5 cm×60 cm column    -   buffer: 200 mM NaCl, 1 mM EDTA, 20 mM Tris-Cl (pH 8.0) and 5%        glycerol    -   flow rate: 1 ml/min    -   procedure        -   equilibrated the column with binding buffer        -   applied the sample to the column and started to collect 20            ml of each fraction        -   eluted the column with 1 bed volume of the buffer        -   regenerated the resin by manufacturer's protocol

This method is difficult from a regulatory standpoint because Con A,which is toxic is leached from the column matrix. In contrast, thepresent method (see Example 16 and Example 38 [Gibson: renumber these?])uses borate to eliminate the glcoprotein. Borate binds to theglycoprotein vicyl hydroxyl groups and imparts a negative charge thusmaking the glycoprotein stick tighter to the anion exchange column.However the rIT as no carbohydrate groups and is eluted by the borate.

Example 10 Construction of Expression Vectors pPGAP-Arg and pPGAP-His

The promoter for Pichia pastoris glyceraldehydes-3-phosphatedehydrogenase gene (P_(GAP)) has been characterized and used forheterologous protein expression in Pichia pastoris (Waterham et al.,1997). P_(GAP) is a strong and constitutive promoter. It was reportedthat protein expression under control of P_(GAP) in glucose-grown Pichiapastoris was higher than that of the commonly used P_(AOX1) inmethanol-grown cells (Waterham et al., 1997; Döring et al., 1998). Thedisadvantage of constitutive promoters in heterologous proteinexpression is that they are not suitable for proteins that are toxic tothe expressing host. Since the EF-2 mutants of Pichia pastoris wereresistant to cytosolic expression of DT A, these mutants should allowconstitutive expression of DT or PE based immunotoxins in their cells.Therefore P_(GAP) was used to drive the expression of A-dmDT390-bisFv inPichia pastoris in the hope that the P_(GAP) would increase theexpression level of protein.

The construct pPGAPArg-A-dmDT390-bisFv was made by replacing the AOX1promoter of pBLARG-A-dmDT390-bisFv with P_(GAP) (FIG. 9 b). First,P_(GAP) was amplified from the expression vector pGAPZ A (Invitrogen) byPCR with primers containing sequences of P_(GAP) 5′ and 3′ ends. The 5′and 3′ end primers had a Nhe I and Hind III added respectively. Afterdigestion with Nhe I and Hind III, the PCR products of P_(GAP) were theninserted in pBLARG-A-dmDT390-bisFv that had been cut with these tworestriction enzymes to remove the AOX1 promoter. The constructpPGAPHis-A-chnDT390-bisFv (FIG. 9 c) was created by joining DNAfragments from plasmids pPIC9K (Invitrogen) andpPGAPArg-A-dmDT390-bisFv. The plasmid pPIC9K was first cut by Sfu I,after filling in with Klenow Fragment by Not I, then the DNA fragmentswere separated by agarose gel electrophoresis. The 5.1 kbp fragmentcontaining kanamycin resistant gene, HIS4 gene and 3′ AOX1 transcriptiontermination (TT) was isolated and ligated with the plasmid DNApPGAPArg-A-dmDT390-bisFv that had been digested with Not 1 and Sca I toremove the 3′AOX1 TT and ARG4 gene.

Example 11 Expression of the Bivalent Immunotoxin Under the Control ofP_(GAP)

As done for the expression under AOX1 promoter, one copy clones wereobtained by transforming mutEF2JC307-8 with constructpPGAPHis-A-c-dmDT390-bisFv; two copy clones by transformingmutEF2JC303-5 with both pPGAPArg-A-dmDT390-bisFv andpPGAPHis-A-dmDT390-bisFv. This time the two copy clones were constructedby two steps. First, mutEF2JC303-5 was transformed withpPGAPArg-A-dmDT390-bisFv, after selection and protein expressionanalysis. The clone that produced the intact immunotoxin at highestlevel was then transformed with pPGAPHis-A-dmDT390-bisFv.

Small scale protein expression was carried out by inoculating a singlecolony to 2 ml YPD, and after overnight growth, cells were seeded in 2ml expression medium at an OD_(600 nm)=0.5, and then incubated at 28° C.for 24 hours before the culture supernatant was analyzed for expressionof the immunotoxin. The expression medium is the similar to BMMYC usedfor expression of the immunotoxin under P_(AOX1), but instead of 0.5%methanol it contains 2% glucose. SDS-PAGE analysis showed thataccwnulation of the intact protein in the culture supernatant of 2 copyclones was slightly higher than that of 1 copy clones. One of the 2 copyclones (Pgap2-9) has consistently producing 10 to 15 μg of intactprotein per ml of culture medium. The results of Western blottinganalysis of culture supernatant and extract cell pellet were consistentwith those obtain from the expression under P_(Aox1).

The production of the bivalent immunotoxin under control of P_(GAP) wasslightly higher than that under P_(AOX1) in shake tube culture. Sincefermentation allowed cells to grow to very high density, the increase inproduction under control of P_(GAP) may be more significant when theproduction is in a bioreactor. The other advantage of P_(GAP) controlledexpression is that production procedure wss simpler and shorter. It didnot require addition and maintenance of methanol in the expressionmedium. The whole production procedure was about 40 hours compared tomore than 72 hour for that of the P_(AOX1) controlled expression.

Example 12 Yeast Strains and Strain Maintenance

In order to optimize fermentation conditions, genetically engineeredPichia pastoris strain JW102 (former name was pJHW #2) was used, whichwas generated for production of the bivalent immunotoxin from the hoststrain GS 115 (Invitrogen, Carlsbad, Calif.) (Woo et al., 2002). TheAOX1 (alcohol oxidase 1) promoter controlled the expression ofimmunotoxin during methanol induction. The gene product was secreted bythe alpha-prepro leader sequence. To compare the growth profile andfermentation parameters in the fermentor, X-33 and JW103 (MutS) ormutEF2JC307-8(2) were used (Table 2) and elsewhere.

TABLE 2 The Pichia pastoris strains used in this study. Names Protein ofinterest Phenotypes JW102* Secretion of bivalent immunotoxin His⁺ Mut⁺JW103* Secretion of bivalent immunotoxin His⁺ Mut^(S) C-4 Cytosolicexpression of A chain of DT His⁺ Mut⁺ X-33 Host strain His⁺ Mut⁺ *JW102and JW103 were renamed from pJHW#2 and pJHW#3, respectively (Woo et al.,2002)

Strain JW102, expressing the bivalent immunotoxin, was genetically verystable. After subculturing the strain more than 60 times onto YPD plates(1% yeast extract, 2% Bacto peptone, 2% dextrose and 2% agar), thestrain maintained expression of the bivalent immunotoxin. A colonyisolated at the very early stage was expanded in YPD broth (1% yeastextract, 2% Bacto peptone, 2% dextrose) and then kept as frozen stock at−80° C. Frozen stock was prepared by mixing a 2-day incubation culturewith an equal volume of 25% (v/v) glycerol and 1 ml of the mixture wasdispensed into a 2 ml Cryo vial.

Example 13 Fermentation

A BioFlo 4500 fermentor (New Brunswick Scientific Company, Edison,N.J.), with a methanol sensor and controller (Raven BiotechnologyCompany, Canada) that maintained methanol at 0.15% (v/v) duringinduction was used. This fermentor was linked to a computer running anAFS-BioConimand Windows-based software (New Brunswick ScientificCompany), which allowed for the control of all parameters by programmedprocesses. The basic initial fermentation medium (10 liters) contained2% (20 g/L) yeast extract, 2% (20 g/L) Soytone Peptone (Difco), 4% (40g/L) glycerol, 1.34% (13.4 g/L) yeast nitrogen base with ammoniumsulfate andwithout amino acids, 0.43% (4.3 mL) PTM1 salt solution and0.02% (v/v) antifoam 289 (Sigma Co.). The PTM1 salt solution(Invitrogen) contained of 24.0 mM (6 g/L) cupric sulfate (CuSO₄.5H₂O),0.534 mM (80 mg/L) sodium iodide (NaI), 17.8 mM (338.6 mg/L) manganesesulfate (MnSO₄.5H₂O), 0.827 mM (200 mg/L) sodium molybdate(NaMoO₄.2H₂O), 0.323 mM (20 mg/L) boric acid (H₃BO₃), 2.1 mM (500 mg/L)cobalt chloride (CoCl₂.6H₂O), 147.0 mM (20 g/L) zinc chloride (ZnCl₂),234.0 mM (65.1 g/L) ferrous sulfate (FeSO₄.7H₂O), 1.64 mM (400 mg/L)biotin, 188.0 mM (18.4 g/L) sulfuric acid (H₂SO₄).

The glycerol batch phase was completed within 18 h of inoculation, andcomplete consumption of glycerol in the culture was detected bymonitoring the DO spike. A glycerol-fed batch phase ensued, during which75% (v/v) glycerol was fed by ramping up the feeding rate at 0.1 g/minto 3.0 g/min for 7 h. Seventy-five percent (v/v) glycerol solutioncontaining 18 ml/L (1.8%) of PTM1 salt solution was used for obtain thedesired cell density for 7 h before methanol induction. Induction wasperformed with a continuous feed of methanol or 4:1 methanol:glycerol(based on volume) with or without 101 mM PMSF (phenylmethylsulfonylfluoride). The feeding rate of methanol or 4:1 methanol:glycerol wasautomatically controlled to be maintained at the set point (0.15% (v/v)methanol in the culture) by the methanol sensor and controller. Themethanol consumption rate was measured by weighing a methanol solutionor methanol/glycerol mixed solution every one minute on a computerinterfaced balance (PG5002S, Mettler Toledo, Switzerland). PMSF wasadded to 1 mM final concentration just prior to induction when PMSF wasadded during methanol induction. A casamino acids or yeast extractsolution (10%, w/v) was fed continuously at 10 ml/h/10 L initial volumeduring methanol induction.

Alternatively, with the EF-2 mutant, the carbon source may be limited tomethanol during induction and the methanol feed rate may be limited toabout 0.5˜0.75 nal/min or lower and regulated by a precision pump (Table3). In run #53, methanol was fully fed by a pump that was controlled bya methanol sensor to maintain a set point of 0.15% methanol in theculture. In run #56, methanol feeding during methanol induction waslimited to 0.75 ml/min. Concentration of bivalent immunotoxin in thesupernatants taken at various induction time points was determined onCoomassie-stained SDS-polyacrylamide gels. For further comparisonbetween both runs, protein yield of the Butyl 650M HIC capture step wasdeter fined from 1 liter of each supernatant. Limited feeding ofmethanol during methanol induction increased the secretion level ofbivalent immunotoxin up to 50 mg/L.

TABLE 3 Limited feeding of methanol at a rate of 0.75 ml/min duringmethanol induction increased secretion level of bivalent immunotoxin inthe EF-2 mutant strain. Run #53 Run #56 Induction Full feeding Limitedfeeding time Purification step of methanol of methanol 22 hr Supernatant12.5 mg/L 15.0 mg/L Butyl 650M HIC 11.7 mg 14.4 mg (from 1 Lsupernatant) 44 hr Supernatant 30.0 mg/L 35.0 mg/L Butyl 650M HIC 23.4mg 28.8 mg (from 1 L supernatant) 67 hr Supernatant 35.0 mg/L 50.0 mg/LButyl 650M HIC 29.3 mg 40.3 mg (from 1 L supernatant)

In order to prepare a seed culture for the fermentor, 50 ml of YSG broth(1% (w/v) yeast extract, 2% (w/v) Soytone Peptone, 1% (w/v) glycerol)was inoculated with 1 ml of a frozen stock (−80° C. in 25% (v/v)glycerol) and then cultivated for 2 days at 28° C. at 250 RPM (orbitdiameter, 1.9 cm).). Thirty ml from a 50 ml culture was used as thefirst seed culture for inoculating 600 ml of YSG broth in two 1 Lflasks. After cultivation for 1 day at 28° C. at 250 rpm (orbitdiameter, 1.9 cm), the cultures were used as the second seed culture forinoculation of 10 L of initial complex fermentation medium in thefermentor. All parameters were automatically managed by runningprocesses programmed in the AFS-BioCommand software. The DO level in thefermentor was maintained at >40% for the entire fermentation with O₂supplementation as needed. The pH in the fermentor was kept at 3.5during the growth phase and at 7.0 during the methanol induction phaseby adding 29% (v/v) NH₄OH or 40% (v/v) H₃PO₄. The pH was ramped up from3.5 to 7.0 for 2 h before the initiation of methanol induction. The pHshifting procedure reduced the secretion of contaminant proteins (75 kDaand 35 kDa bands) into the supernatant. The temperature was set at 28°C. for growth and 15-25° C. during methanol induction. The inductiontemperature was ramped down from 28 to 25-15° C. during the first 4 h ofmethanol induction. Reducing the bioreactor agitation may increase thefraction of monomeric and bioactive immunotoxin. A bioreactor agitationof 400 rpm increases the fraction of monomeric and bioactive immunotoxinby 50% over a bioreactor agitation of 800 rpm (FIG. 12). Providing adetergent or other denaturant during agitation may reduce aggregation ofthe immunotoxin. Including TWEEN 20® at 0.01% during agitation ofimmunotoxin further reduces aggregation and increases the fraction ofmonomer and bioactive immunotoxin to 90% (FIG. 13). After harvesting theculture, the supernatant was prepared by centrifugation (2,800×g at 4°C. for 30 min). EDTA was added to a final concentration of 5 mM toprevent protein degradation during storage at 4° C.

Example 14 Measurement of Wet Cell Density (%, w/v) for Monitoring CellGrowth

One ml of culture sample was placed in a tared 1.5-ml microcentrifugetube and spun at 20,800×g at 25° C. for 2 min. The supernatant wasremoved with a pipet and residual liquid in the tube was blotted withfilter paper. After weighing the tube containing the cell pellet, thewet cell density (%, w/v) was calculated.

Example 15 Purification

A scaleable 3-step procedure for purification of the bivalentimmunotoxin has been developed that utilizes borate anion exchangechromatography to eliminate contaminating host glycoproteins.Purifications were performed with 1 L of centrifuged supernatant. Nodialysis or diafiltration step was employed. In brief, 1 L ofsupernatant was mixed with 28.4 g of solid Na₂SO₄ and applied to a 100ml bed of butyl-650M and eluted with 5% glycerol, 20 mM tris and 11n MEDTA, pH 8.0, after washing in 200 mM Na₂SO₄. 600 ml of eluant wasdiluted with 4.2 L of TE buffer (20 mM tris, 1 mM EDTA, pH 8.0) andapplied to a 40 ml bed of Poros 50 HQ. The bivalent immunotoxin waseluted in steps of sodium borate buffer from 25-100 mM, and thenglycoproteins and some highly aggregated immunotoxin were eluted with 1M NaCl. 1.2 L of the borate eluant was diluted with 3.6 L of TE bufferand applied to a 5 ml prepacked bed of Hi-trap Q. After washing, thebivalent immunotoxin was eluted with a 0-400 mM NaCl gradient.

Butyl-650M hydrophobic interaction chromatography (BIC): Approximately100 ml of Butyl-650M resin (Tosoh Biosep LLC) was packed in a 5 cm×20 cmXK column (Amersham Pharmacia Biotech) and the column was equilibratedwith Buffer A containing 200 mM Na2SO4, 1 mM EDTA, 20 mM Tris-Cl buffer(pH 8.0). Solid sodium sulfate and 1 M Tris-Cl buffer (pH 8.0) wereadded to 1 liter of the supernatant to a final concentration of 200 mMand 20 mM, respectively. The sample was filtered with a 802 flutedfilter paper (>15 um particle retention: Whatman Inc.; Clifton, N.J.,USA) before loading. The flow rate was 44 cm/hour (14.4 ml/min). Afterequilibrating the column, 1 L of the prepared sample was applied ontothe column, and then the column was washed with 6 column volumes ofbinding buffer A. The bound proteins to Butyl-650M resin were elutedwith 6 column volumes of Buffer B containing 5% glycerol, 1 mM EDTA, 20mM Tris-Cl buffer (pH 8.0). The eluted fractions having the immunotoxinwere pooled for the next step (volume: ˜600 ml). After each run, thecolumn was regenerated according to the manufacturer's protocol. Allsteps were performed in a cold room except for the first step that wascarried out at room temperature.

Poros 50 HQ anion exchange chromatography (AEX) by step-elution withsodium borate buffer: Approximately 40 ml of Poros 50 HQ resin(PerSeptive Biosystems) was packed in a 2.6 cm×20 cm XK column (AmershamPharmacia Biotech) and then the column was equilibrated with Buffer B.The pooled sample from the previous step was diluted with 4.2 L of TEbuffer (20 mM Tris-Cl, 1 mM EDTA, pH 8.0). The diluted sample was loadedonto the column at a flow rate of 80.2 cm/hour (7.08 ml/min), and thenthe column was washed with 6 column volumes of Buffer B. The boundproteins were eluted in steps of sodium borate of 25 mM, 50 mM, 75 mMand 100 mM in Buffer B (10 column volumes for each step). These elutedfractions were pooled for the next step. The residual protein bound tothe resin was stripped with 6 column volumes of 1 M NaCl in Buffer B.After each run, the column was washed with 0.5 M NaOH and thenre-equilibrated with Buffer B minus 5% glycerol for the next use.

Hi-trap Q anion exchange chromatography: A prepacked Hi-trap Q anionexchange column (5 ml) was purchased from Amersham Pharmacia Biotech.The pooled sample from the previous step was diluted with 3.6 L of TEbuffer. The sample was loaded onto the equilibrated column with Buffer Bat a flow rate of 221.5 cm/hour (7.08 ml/min). The column was washedwith 5 column volumes of Buffer B. The bound immunotoxin was eluted witha linear 0˜400 mM NaCl gradient in Buffer B (20 column volume). The flowrate for washing and eluting steps was 2 ml/min and fraction size was 5ml.

Example 16 Measurement of Protease Activity in the Supernatant

Unnicked CRM9 (one point mutation in the recognition domain ofdiphtheria toxin (7)) was used as the substrate for measurement ofserine-protease activity in the supernatant at a final concentration of225 μg/ml. The supernatant was incubated at 28° C. with shaking at 250rpm (orbit diameter, 1.9 cm) for 20 h before applying to a 4-20%tris-glycine precast SDS-PAGE gel in the presence of reducing agent (100mM dithiothreitol). CRM9 contains a well exposed furin/Kex-2 cleavagesite between the A fragment (22 kDa) and B fragment (40 kDa) spanned bya disulfide bond. Protease activity in the medium was detected by lossof unnicked CRM9 under reducing condition, and the band intensity of theunnicked CRM9 was quantified by densitometry on Coomassie stained gels.

Example 17 SDS-PAGE and Western blotting

Proteins in culture supernatants were subjected to SDS-PAGE utilizingtris-glycine 4˜20% precast gels (Invitrogen) under non-reducing and/orreducing conditions. For Western blotting, the fractionated proteinswere transferred onto nitrocellulose membranes by electroblotting.Non-specific binding was blocked with 5% nonfat skimmed milk in TBSTbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% TWEEN 20®). Goatpolyclonal antibody directed against diphtheria toxin (Thompson et al.,1995) diluted 1:2000 was used as the primary antibody, and alkalinephosphatase-conjugated rabbit anti-goat IgG (Roche MolecularBiochemicals) diluted 1:5000 was used as the secondary antibody. Theimmunotoxin was visualized with one-step NBT/BCIP substrate (PierceChemical Company). Alternatively, rabbit polyclonal antibody directedagainst (G₄S)₃ linker was used as the primary antibody for detectingintact immunotoxin and degraded products since the bivalent immunotoxincontained three (G₄S)₃ linkers. This antibody was raised against thesynthetic peptide, whose amino acid sequence was GGGGSGGGGSGGGGS (SEQ IDNO: 17).

Example 18 Cytotoxicity Assay

The tests to measure the specific cytotoxicity of anti-human anti-CD3immunotoxins expressed in Pichia pastoris were performed as described(Neville et al., 1992). Briefly, immunotoxins were applied to Jurkatcells, a human CD3∈+ T cell leukemia line, (5×104 cells/well) in 96-wellplates in leucine-free RPMI 1640 medium. After 20 hours, a 1 hour pulseof [³H] leucine was given. The cells were then collected onto filterswith a cell harvester. After addition of scintillant, samples werecounted in a Beckman scintillation counter using standard liquidscintillation counting techniques.

Example 19 Measurement of Cell Viability

In order to measure cell viability of cultures taken at various timepoints in fermentation, Ormerod's method was modified (Ormerod, 2000).Fluorescein diacetate (FDA) and propidium iodide (PI) were used as vitaldyes of cell viability. FDA taken up by Pichia pastoris was converted tofluorescein by an intracellular esterase. If a cell has an intact plasmamembrane, fluorescein is retained and PI is excluded. In brief 500 μl ofa suspension of Pichia pastoris cells at 106 cells/ml in the PBS bufferwere mixed with 50 μl of FDA solution (10 μg/ml) and 50 μl of PIsolution (100 μg/ml). After incubation at room temperature for 10 min,cell viability of the sample was analyzed by flow cytometry. The viablecell gate included green fluorescence and excluded red fluorescence.

Example 20 Quantification of Concentration of the Bivalent Immunotoxin

A Superdex 200 10/300 GL prepacked column (dimension 1.0 cm×30 cm) waspurchased from Amersham Pharmacia Biotech. The column was connected toan HPLC system (GBC Scientific Equipment; Arlington Heights, Ill., USA).Gel filtration buffer consisted of 90 mM sodium sulfate(Na₂SO₄), 10 mMsodium phosphate monobasic (NaH₂₇PO₄.H₂O) and 1 mM EDTA (pH 8.0). Theflow rate was 0.5 ml/min and injection volume was 500 Purifiedimmunotoxin of known concentration based on UV absorbance (25) served asa standard.

Quantification of the bivalent immunotoxin in supernatants or liquidsamples, was performed by comparing the intensity of Coomassie-stained4-20% precast SDS gels with that of immunotoxin standards of knownconcentration.

Example 21 Immunotoxin Toxicity During Expression in Pichia pastoris isManifest by a Reduction in AOX1 Activity

The bivalent immunotoxin in Pichia pastoris was expressed via thesecretory route. This secretion of the bivalent immunotoxin in Pichiapastoris significantly attenuated the toxicity of the immunotoxin (Wooet al., 2002), but the bivalent immunotoxin expression depressedmetabolic capacity of methanol utilization and growth reduction duringmethanol induction in fermentor culture.

In the metabolism of methanol by Pichia pastoris, oxidation of methanolby alcohol oxidase (AOX1) is the rate-limiting reaction (Veenhuis etal., 1983), and the amount of the AOX1 gene product determines howrapidly methanol is metabolized. AOX1 can account for 30% of theproteins in Pichia pastoris cells utilizing methanol. Therefore,measurement of methanol consumption rates during methanol inductionreflects the AOX1 level and provides an indication of how the expressionof the bivalent immunotoxin affects protein synthesis and degradation ofAOX1 in Pichia pastoris. To this end, profiles of the methanolconsumption rate in a fermentor culture were compared between the wildtype host strain X-33 and the JW102 strain, which expressed the bivalentimmunotoxin via the secretory route. Under the fermentation conditionswhere casamino acid supplements were used during methanol induction,X-33 had a maximum 1.95 ml/min of methanol consumption at 25° C. and theconsumption rate was maintained at more than 70% of the maximum rateduring the whole methanol induction phase (FIG. 11). For the immunotoxinexpressing strain JW102 (Mut+), the maximum methanol consumption ratewas approximately 1.10 ml/min at 23° C. After the peak point at 7˜8hours following the initiation of methanol induction, the consumptionrate was gradually decreased to 20% of the maximum rate. Within thefirst 18 hours of methanol induction, the methanol consumption ratedropped below 50% of maximum methanol consumption rate (FIG. 11). Theselow levels of methanol consumption were associated with low levels ofwet cell density increase, 2% for JW102 versus 10.5% for X-33 at 44hours (FIG. 14).

Example 22 Use of PMSF and Casamino Acids or Yeast Extract During theMethanol Induction Phase

In the initial stages of fermentation optimization, supplementing ofPMSF and casamino acids during methanol induction was crucial forboosting the expression level in the fermentor. Without these twocomponents during methanol induction, the expression level of thebivalent immunotoxin reached a maximum 7 hours after initiation ofmethanol induction and then decreased. However, supplementing these twocomponents during methanol induction extended the optimal induction timefrom 7˜8 hours to 24˜48 hours after the start of methanol induction. Inaddition, the expression level was improved up to 2-fold.

To avoid the use of animal-derived material, yeast extract wassubstituted for casamino acids. This change resulted in a substantialincrease in the expression level by 30% and in wet cell density by 45%.Gain of wet cell density for JW102 by continuous feeding of yeastextract was close to that for X-33 during methanol induction (FIG. 14).These improvements were due to constancy of the methanol consumptionrate at greater than 80% of the maximum rate (FIG. 11).

An example of the final expression method, disclosed herein (seeExample_(—)41), uses the toxin resistant EF-2 mutant, limited methanolfeeding during induction of 0.5 to 0.75 ml/min (per 10 L initial medium)without an additional carbon source, extension of induction time to 163h, a temperature of 15° C., a continuous infusion of yeast extract,limitation of agitation speed to 400 RPM, addition of antifoam agent upto 0.07%, and supplementation of oxygen when DO levels fall below 40%.Under these conditions PMSF and Casamino acids are not required.Casamino acids are an animal product and are frowned upon by the FDA.PMSF, a protease inhibitor aided to prevent product breakdown, is toxicand requires additional documentation of its absence from the finalproduct, so these changes aid regulatory approval. Using thismethodology the yield is 120 mg/L (see example 41).

Example 23 Use of Methanol/Glycerol Mixed Feed During Methanol Induction

The expression level of the bivalent immunotoxin was positively relatedto the gain of wet cell density during the first 44 hours of methanolinduction. In low-producing cultures, the gain of wet cell density wasless than 6.0%. However, in fermentation runs producing more than 25mg/L of the bivalent immunotoxin, the gain of wet cell density (%)during the first 44 hours of methanol induction was an average of 9.26%(FIG. 14). The gain of wet cell density during methanol induction washard to achieve without continuous feeding of glycerol as the additionalcarbon source. Therefore a methanol/glycerol (4:1) mixed feed was usedto support cell growth during methanol induction.

Wild type strain X-33 did not produce immunotoxin (FIG. 21A) and servedas a control for monitoring methanol consumption and cell growth. Thisstrain had a maximum methanol consumption of 1.95 ml/min at 25° C. Thisconsumption rate was maintained at >70% of the maximum rate for theentire methanol induction phase (FIG. 21A). The wet cell densityincreased continuously during the 44 h methanol induction. TheDT-resistant immunotoxin producing EF-2 mutant strain, mutEF2JC307-8(2)was used as another control for comparing methanol consumption and cellgrowth upon the secretion of immunotoxin. This EF-2 mutant strain hadsimilar profiles of methanol consumption and wet cell growth to those ofwild type strain X-33 during induction (FIG. 21A). The maximum methanolconsumption rate and wet cell gain during 44 h of methanol inductionwere 2.2 ml/min and 9.17%, respectively. However, the use of the EF-2mutant did not improve immunotoxin secretion under the fermentationconditions for the JW102 strain producing immunotoxin. For strain JW102, the maximum methanol consumption rate was 1.30 ml/min at 25° C.After peaking at 7-8 h following the initiation of methanol induction,the consumption rate decreased to 15% of the maximum rate at 44 h ofmethanol induction. Within the first 22 h of methanol induction, themethanol consumption rate dropped to <50% of the maximum methanolconsumption rate (FIG. 21B). This low level of methanol consumptionresulted in less increase in wet cell density, 2.0%, for JW102 than forX-33 (10.5%) (FIGS. 21A and 21B). There was little or no increase in wetcell density after the first 22 h of methanol induction and the secretedlevel of immunotoxin decreased from 15 to 10 mg/L. Immunotoxin breakdownproducts were not detectable on the SDS gels used to monitor productstability.

Example 24 Yeast Extract Feeding, Methanol Consumption, and ImmunotoxinProduction

The decreased methanol consumption and cell growth rate associated withimmunotoxin production can be due to the toxicity of the immunotoxin toP. pastoris. If yeast extract was fed continuously to the bioreactorwith methanol as the sole carbon source (FIG. 21C), then peak methanolconsumption was less than with the wild type strain and the EF-2 mutantstrain (FIG. 21A), but the decrease after 10 h was eliminated and cellgrowth increased throughout the induction period. This growth responsewas coupled with a loss of immunotoxin in the medium after 8 h,indicating protease activity. Immunotoxin fragments were present at 4 hafter induction, and no intact immunotoxin was detected by 19 h afterinduction (FIG. 22A). If the medium collected at various time points wasincubated with purified immunotoxin, then the amount of immunotoxinfragments formed depend on the age of the medium (FIG. 22B). Forexample, at 49 h post induction the intact immunotoxin band is greatlyreduced and the 36.5 kDa band representing degraded fragments is greatlyincreased relative to samples from earlier time points.

The reduction in methanol utilization that was corrected by yeastextract feeding (FIG. 21C) is apparently secondary to inhibition ofprotein synthesis by the immunotoxin following ADP-ribosylation of EF-2.This was shown by the fact that a P. pastoris strain producingimmunotoxin and engineered to toxin resistance in the EF-2 gene (13)consumed methanol at the wild type strain rate (FIG. 21 and FIG. 21legend). In the toxin-sensitive strain, inhibition of protein synthesiscan occur if the immunotoxin gains access to the cytosol compartmentwhere EF-2 resides. Two distinct mechanisms can produce this effect. Onemechanism is post-translational translocation where the entireimmunotoxin is translated before entering the Sec61 translocon (16).This would provide a brief opportunity for ADP ribosylation of EF-2.Post-translational translocation is common when the signal peptide isalpha mating factor as it is in this case (24). Another mechanism is thewell documented proton mediated catalytic domain translocation across aninternal membrane compartment (2). This can occur from the mildly acidicGolgi compartment or the more acidic vacuole. Whichever immunotoxintranslocation mechanism is dominant, yeast extract feeding eitherinterferes with this step, or with the subsequent ADP-ribosylation ofEF-2 either directly or by attenuating the catalytic activity of thetranslocated toxin A chain

Example 25 Addition of Glycerol to the Methanol Feed with Yeast ExtractFeeding

The protease activity observed when methanol was the sole carbon sourcecould be a result of leaking from dead or injured cells. When a 4:1methanol:glycerol feed (FIG. 21D) was substituted for the pure methanol(FIG. 21C) the level of immunotoxin in the medium rose to 20 mg/L at 44h. Only minimal degradation products now could be detected in SDS gelsof proteins in the medium (FIG. 23, far right panel). Themethanol-glycerol mixed feed without yeast extract could not sustain themethanol consumption or the continual increase in cell mass, and thefinal immunotoxin production was to 15 mg/L (FIG. 21E).

Although continuous feeding of yeast extract largely corrected thereduction in methanol metabolism, immunotoxin production was low and wasassociated with extensive proteolysis (FIGS. 21C and 22). This extensiveproteolysis was reversed by providing supplemental carbon in the form ofa mixed 4:1 methanol:glycerol feed (FIG. 21D and FIG. 23, panel 23° C.),which increased immunotoxin production to 20 mg/L. It has been reportedthat there is an optimal maximal specific growth rate during P. pastorismethanol fed-batch culture, which when exceeded depresses heterologousprotein production (27). Feeding methanol at the optimal rate and addingglycerol at a rate of 20% of the maximal glycerol growth rate increasedheterologous protein production by 50% (27). This increase can resultfrom increased metabolism of formaldehyde and H₂O₂ and higher activityof catalase and AOX. In the case of secreted proteins these metabolicchanges also can reduce the amount of excreted proteases and reduce thenumber of dead or injured cells leaking proteolytic enzymes.

Example 26 Low Temperature and Secretion of Bivalent Immunotoxin

Low temperature can improve the yield of heterologous protein expressionin P. pastoris either by enhancing protein folding within the ER and/orby reducing medium protease activity (9). At 15° C. methanol consumptionat 44 h was reduced by 25%, however cell growth was maintained (FIG.15C). Immunotoxin production increased by 50% at 44 h (30±0 mg/L, n=3)and almost 100% at 67 h (37±2.9 mg/L, n=3). Most of the increase inimmunotoxin secretion occurred between 20-15° C.

The highest expression level was observed at 17.5° C. (FIG. 15A), butthe final yield obtained by the 3-step purification procedure of theimmunotoxin was the highest at 15° C. (FIG. 15B) and averaged 13.8±1mg/L (n=3) and 16.0±1 mg/L (n=3) at 44 and 67 h, respectively. Thepurified immunotoxin produced at 15° C. was fully functional, asconfirmed by measuring specific T cell cytotoxicity in protein synthesisassay yielding IC₅₀ values for three individual production runs of 1.2(±0.1)×10-13 M compared to 2×10-13 M for the average of three runs fromshake flask culture.

The amount of degraded immunotoxin bands noted on SDS gels frombioreactor supernatants (all receiving continuous 10 mM PMSF feeding)were reduced from modest levels at 23° C. to undetectable levels at 15°C. (FIG. 23). By using a sensitive assay for serine Ibex-2-likeproteases employing a mutant diphtheria toxin (CRM9) substrate, proteaseactivity was undetectable at 67 h at 15° C. although activity wasdetected at 67 h when PMSF was not infused (FIG. 24). At 15° C. gelpatterns and immunotoxin yields were identical whether or not PMSF wasinfused.

Cell viability of 15° C. bioreactor samples from the methanol-glycerolmixed feed plus yeast extract medium assayed by flow cytometery had alow level of dead cells: 0.7±0.22% (confidence limit 99%) glycerolfed-batch phase; glycerol-methanol mixed feed, 1.2±0.58% (confidencelimit 99%) at 22 h, 1.7±0.61% (confidence limit 99%) at 44 h and1.1±0.51% (confidence limit 99%) at 67 h (the dead cell fraction wasdetermined from one fermentation run). The viable cells showingintracellular esterase activity were present in over 96% of the cells atall time points during methanol induction.

Lowering the induction temperature from 23-25° C. to 15° C. furtherincreased the immunotoxin level to 30 mg/L at 44 h and 37 mg/L at 67 h(FIG. 21F). Low induction temperature was associated with a low andconstant level of dead cells during induction (<2.0%) and reducedprotease activity toward immunotoxin within the bioreactor even thoughsmall amounts of protease activity could be detected by a sensitiveassay (FIGS. 23 and 24). These results are consistent with a studyutilizing temperature limited (12° C.) fed-batch technique (9). In thetemperature limited fed-batch technique, dead cells were reduced from 9%to <1% at 44 h compared to a methanol limited fed-batch process at 30°C. This reduction in dead cells was associated with a marked reductionin degraded product (lipase) and a 2-fold increase in intact product atlate time points. These changes were attributed to the avoidance ofoxygen deprivation at high cell densities. AOX activity increased morethan 2-fold at 67 h in the temperature-limited fed-batch technique

Lowering induction temperature can also result in increased immunotoxinsecretion by the balancing of immunotoxin input and output through thesecretory pathway by reducing the overall protein synthesis rate. In theexpression and secretion of heterologous proteins, each protein appearsto have an optimal secretion level. Expression beyond the optimal level(overexpression) can reduce secreted protein yields (1, 11, 13, 15). Thebivalent immunotoxin also can require a longer processing time forcorrect folding because of the multi-domain structure of this protein,which has low activity after in vitro refolding following expression inE. coli (25). The methanol consumption rate was reduced by only 25% ingoing from 23° C. to 15° C. and the cell growth rate was unchanged at 44h.

Example 27 Complex Media for Production of Bivalent Immunotoxin inPichia pastoris

The uses of the complex components in the initial fermentation mediawere necessary to obtain a reasonable range of the expression level ofthe bivalent immunotoxin in the fermentor. In the initial fermentationruns, very low production of the bivalent immunotoxin in the fermentorwas observed when the standard defined medium was used. Therefore,Soytone Peptone and yeast extract-based medium was developed containing4% glycerol, 2% yeast extract, 2% Soytone Peptone, 1.34% yeast nitrogenbase with ammonium sulfate andwithout amino acids, 0.43% PTM1 saltssolution and 0.01% antifoam 289.

Example 28 Mut+ Versus MutS Phenotype

Different Mut (methanol utilization) phenotype strains derived fromPichia pastoris GS115 (Mut+) and KM71 (MutS) were tested to compare theexpression level of the bivalent immunotoxin in the fermentor. In thefermentor, the MutS phenotype strain has advantages, such as easycontrol of induction temperature, no need to supply pure oxygen, andresistance to a high concentration of methanol. Although these twodifferent phenotype strains did not make a difference in the expressionlevel in test tube culture, the expression level of the Mut+ strain inthe fermentor was 5˜7-fold higher than that of the MutS strain.

Example 29 pH Shifting Procedure Reduces Contaminant Proteins in theSupernatant

There was a great difference between shake flask culture and fermentorculture for the expression of the bivalent immunotoxin. In shake flaskculture, it is possible to replace the culture medium with freshinduction medium, resulting in removal of cell membrane fragments, DNAand proteases derived from cell lysis during the growth period andproteins secreted by Pichia pastoris. However, those moleculesaccumulate for the whole period of fermentation and they are oftenproblematic in the purification process.

In order to reduce this kind of problem in the fermentor, a pH shiftingprocedure was employed. Pichia pastoris can normally grow within therange of pH 3˜7. Pichia pastoris was cultivated at a low pH such as pH3.5 during the glycerol batch phase and the glycerol-fed batch phase,and induced at pH 7.0 for production of the bivalent immunotoxin. The pHshifting procedure provided the supernatant with the dominant bivalentimmunotoxin, because the amount of secreted proteins in Pichia pastoriswas significantly decreased at low pH even though the expression levelof the bivalent immunotoxin was not affected.

Example 30 The Use of Glucose for Tight Control of the AOX1 Promoter

In general, tight gene control is necessary to obtain toxic proteins inhost cells. The expression of the bivalent immunotoxin was toxic toPichia pastoris. Since the AOX1 promoter cannot tightly control geneexpression in the presence of glycerol as the carbon source, thebivalent immunotoxin was observed before methanol induction on Coomassiestained SDS-polyacrylamide gels. The glycerol-fed batch phase wasreplaced with a glucose-fed batch phase for tight gene control, becauseglucose represses AOX1-driven gene expression (Tschopp et al., 1987).However, the replacement of glycerol with glucose in the fed batch phasedid not change the final expression level of the bivalent immunotoxin.Glycerol was used during the fed batch phase because glucose took timeto dissolve at a high concentration. When combined with the glycerol-fedbatch phase, the pH shifting procedure prevented the appearance of thebivalent immunotoxin on Coomassie-stained SDS-polyacrylamide gels duringthe glycerol-fed batch.

Example 31 Optimal pH for Expression of the Bivalent Immunotoxin

In order to determine optimal pH for the expression of the bivalentimmunotoxin, the expression strain JW 102 was induced for 24 hours inthe range of pH 3.5 to 8.0 in test tube cultures, and the bivalentimmunotoxin in the supernatants was compared on a Coomassie-stainedSDS-polyacrylamide gel and Western blotting. Sodium citrate buffer (pH3.5˜5.5), bis-tris buffer (pH 6.0˜7.0) and tris buffer (pH 7.5˜8.0) wereused for maintenance of the cultures at the indicated pH.Simultaneously, colony forming units in the cultures at the end ofmethanol induction were measured as previously described (Woo et al.,2002). Below pH 6.0, the bivalent immunotoxin was not detectable onWestern blots. Although the Western blot shows similar expression levelsin the range of pH 6 to 8, the Coomassie-stained SDS-polyacrylamide gelindicates pH 7.0 was the optimum pH level for the expression of thebivalent immunotoxin. Pichia pastoris had similar colony forming unitsin the range of pH 3.5 to 7.0, but the colony forming units were sharplydecreased at above pH 7.4. Since pH 7.4 was the upper edge of optimal pHrange, the expression level at pH 6.7 was also tested in the fermentor.However, there was no difference in the expression level at pH 6.7 and7.4.

Example 32 Reproducibility and Cell Viability of Optimized FermentationRuns

Under the optimized fermentation conditions, the expression level of thebivalent immunotoxin increased to 40 mg/L at 67 hours of methanolinduction (FIG. 16). The expression levels in the supernatants at 44hours and 67 hours of methanol induction, and the final yield obtainedby the 3-step purification procedure for the bivalent immunotoxin werereproducible. As shown in Table 4, very similar levels of bivalentimmunotoxin were obtained in 3 independent fermentation runs under theoptimized conditions. More importantly, the final yields of the purifiedbivalent immunotoxin were very similar to each other, indicating thatproduced supernatants had similar quality of the bivalent immunotoxin.Under the optimized fermentation conditions, cell viability duringmethanol induction phase was maintained at greater than 95% asdetermined by flow cytometry.

TABLE 4 Reproducibility of optimized fermentation condition¹ andpurification². Purified Methanol immunotoxin induction Expression from 1L time level supernatant Run no. (hrs) (mg/L) (mg) 1 44 30 16 67 40 18 244 30 16 67 40 18 3 44 30 16 67 40 18 ¹Optimized condition: inductiontemperature at 15° C.; continuous feeding of 10% yeast extract feedingat 8.95 ml/hr; methanol/glycerol (4:1) mixed feed for methanolinduction. ²For purification of the bivalent immunotoxin, a 3-stepprocedure (Woo and Neville, 2003) was used.

Example 33 Relationship Between Induction Time Aid Formation of theAggregates

Immunotoxin aggregates were accumulated in the supernatant duringinduction. In order to determine the relationship between induction timeand aggregate formation, fractionated samples were taken at 22, 44 and67 hours of methanol induction by a Superdex 200 gel filtration and thenanalyzed fractionated samples on SDS-PAGE gels. The 22, 44 and 67 hoursamples contained 50.0, 60.0 and 66.7% of dimeric and higher oligomericforms of the immunotoxin. These aggregate forms of the immunotoxin hadonly 10% specific toxicity of the monomeric immunotoxin to Jurkat cells.

In addition, the accumulation of immunotoxin aggregates significantlyreduced bioactivity of the supernatant. However, bioactivity wasrecovered by the butyl 650M capturing step developed in a previousstudy. This result suggested the possibility that some portion ofimmunotoxin aggregates were reversible.

The use of antifoam agents at a concentration above 0.01% reducedformation of aggregates. These immunotoxin aggregates did not bind wellin thiophilic adsorption used as the capture step before developing a3-step purification procedure. In the initial stages of fermentationoptimization, antifoam agents were used at the minimum concentrationthat could control excessive foaming in the fermentor. However, morethan 50% of the bivalent immunotoxin was lost at the first capturingstep when antifoam 289 was used at 0.005% in the initial fermentationmedium. The use of antifoam 289 at a concentration of more than 0.01% inthe initial fermentation medium was crucial to obtain reasonable yieldsof more than 90% in the first capture step.

Example 34 Protein Quantification by Comparison on SDS-PAGE andCytotoxicity Assay

The concentration of the immunotoxin was quantified by SDS-PAGE using animmunotoxin standard of known concentration prepared previously (Woo etal., 2002). Samples to be measured were subjected to SDS-PAGE utilizingtris-glycine 4-20% precast gels (Invitrogen) under non-reducing orreducing conditions.

The specific cytotoxicity of the purified anti-human anti-CD3immunotoxins were performed as described (Neville et al., 1992).Briefly, immunotoxins were applied to Jurkat cells, a human CD3∈+ T cellleukemia line, (5×104 cells/well) in 96-well plates in leucine-free RPMI1640 medium. After 20 hours, a 1 hour pulse of [3H] leucine was given.Cells were collected onto filters with a Skatron harvester.

After addition of scintillant, samples were counted in a Beckmanscintillation counter using standard LSC techniques.

Example 35 Butyl 650M Hydrophobic interaction Chromatography (Butyl 650MHIC)

As shown in FIG. 17, Butyl 650M HIC was an efficient capture step forimmunotoxin in supernatant. However, glycoproteins were also purifiedwith the immunotoxin during this step. Among these glycoproteins,identified by periodic acid Schiff staining, the glycoprotein species ofapproximately 45 kDa (arrow in FIG. 17) impeded isolation of the pureimmunotoxin. By conventional chromatography such as gel filtration andanion exchange chromatography, these glycoproteins were not separatedfrom the immunotoxin, indicating that these 45 kDa glycoprotein specieswere present in dimeric form and had similar isoelectric points.Therefore these 45 kDa glycoproteins were very similar to theimmunotoxin in size and isoelectric point as well as in hydrophobicity.

Various hydrophobic resins which complied with GMPs (Good ManufacturingPractices) were evaluated. Among these resins, Butyl 650M appeared tohave the best binding and eluting profile of the immunotoxin. Otherhydrophobic resins may be used in the present invention. Also it wasfound that 200 mM of sodium sulfate was a suitable concentration forbinding of the immunotoxin to the butyl 650M resin.

The fermentor culture normally had approximately 30% of wet cell densityat the end of the fermentation run. In large-scale production, thesupernatant is obtained by continuous centrifugation requiring a 3-folddilution of the high cell density culture. The immunotoxin in thediluted sample was processed the same as the immunotoxin in thesupernatant which was effectively bound to the Butyl 650M resin at 200mM sodium sulfate.

Example 36 Poros 50 HQ Anion Exchange Chromatography by Step-Elutingwith Sodium Borate Buffer

By employing borate anion exchange chromatography, the immunotoxin wassuccessfully separated from the Pichia pastoris glycoproteins (FIG. 18).The immunotoxin was bound to anion resin by diluting the sample from theprevious step, simplifying the purification procedure. In fractionseluted with 50 mM, 75 mM and 100 mM sodium borate in Buffer B (lane 9,10, 11 in FIG. 18), most of the immunotoxin was present in monomericform. These 3 fractions were pooled for the next step.

In order to remove glycoprotein species in the sample obtained from theprevious step, sodium borate in anion exchange chromatography was used,because sodium borate increases the negative charge of glycoproteins bybinding to the carbohydrate residues of the glycoproteins. Theimmunotoxin binds to anion exchange resins at pH 8.0 (Woo et al., 2002).Preliminary experiments were designed for optimizing binding conditionsof the immunotoxin in the presence of sodium borate. Aliquots of thedialyzed sample against Buffer B were mixed with different volumes of200 mM sodium borate in Buffer B to obtain the designated concentrationof sodium borate. The prepared samples were then loaded onto a Poros 50HQ anion column (40 ml) equilibrated with Buffer B containing acorresponding concentration of sodium borate. At 100 mM of sodium boratethe immunotoxin did not bind to the Poros 50 HQ anion resin, but themajority of glycoproteins still bound. At a concentration of sodiumborate below 50 mM, the immunotoxin bound to the Poros 50 HQ anionresin.

Conditions of step elution were further analyzed with sodium borateafter binding of the immunotoxin to an anion exchange column. First, thesample dialyzed against Buffer B was bound to the anion column and theneluted in steps of increasing concentration of sodium borate (100, 120,140, 200 mM) and 1 M NaCl. The bound immunotoxin was mainly eluted at100 mM sodium borate, but these eluted fractions also containedsignificant amounts of 45 kDa glycoproteins which were not separable inthe next step. The majority of glycoproteins were eluted at 1 M NaCl.After loading the same sample as the first experiment, the boundimmunotoxin was eluted in steps of 50, 75 and 100 mM sodium borate and 1M NaCl. A majority of the bound immunotoxin was eluted at 75 mM sodiumborate. However, a protein band corresponding to 21 kDa was included inthe fraction eluted with 50 mM sodium borate. After binding to thecolumn, the bound immunotoxin was eluted in steps of 25, 50, 75 and 100mM sodium borate and 1 M NaCl in Buffer B (FIG. 18). By washing with 25mM sodium borate buffer, the amount of a protein band corresponding to21 kDa was reduced.

Example 37 Comparison with Phenylboronate Affinity Chromatography

In order to compare separation profiles, phenylboronate affinitychromatography was performed. The eluant from the butyl 650M HTC capturestep was dialyzed against the low ionic strength buffer (10 mM HEPES,0.25 mM EDTA and 20 mM MgCl2, pH 8.2) for phenylboronate affinitychromatography. The dialysed sample was applied to a 5 ml bed volumecolumn of phenylboronate agarose (Sigma Co.), washed with the samebuffer, and then the bound proteins were eluted with either 0-100 Msodium borate gradient or 0-50 mM sorbitol gradient in the same buffer(20 bed volumes). Glycoproteins and the immunotoxin were bound underbinding condition of low ionic strength. The glycoproteins andimmunotoxin were not separated by phenylboronate affinitychromatography. The glycoproteins and immunotoxin were co-eluted witheither 0-100 mM sodium borate gradient or 0-50 mM sorbitol gradient.

Example 38 Q Anion Exchange Chromatography

Q anion exchange chromatography was used for concentration of thediluted sample that was obtained from the Poros 50 HQ anion exchangechromatography. At a concentration of sodium borate below 50 mM theimmunotoxin was bound to the anion exchange resin. Accordingly, thepooled sample from the previous step was diluted with 3 sample volumesof TE buffer (20 mM Tris-Cl and 1 mM EDTA, pH 8.0), resulting in lessthan 20 mM of sodium borate in the diluted sample. As expected, theimmunotoxin was effectively bound to the Q anion exchange column. Thebound immunotoxin was eluted with 0˜400 mM NaCl gradient elution (20column volumes). The immunotoxin fractions were pooled and then assessedfor yield, purity and toxicity of the final preparation by SDS-PAGE andprotein synthesis assay.

Example 39 Protein Yield, Repeatability of Purification Procedure,Purity and Function of the Purified Immunotoxin

Table 4 summarizes the immunotoxin yields which were obtained in 3batches of the 3-step purification runs by using the supernatants takenat 44 hours of methanol induction from 3 fermentation runs which werecarried out under relatively similar fermentation conditions and hadsimilar expression levels of the immunotoxin. The average yield of thispurification batch was 52.8%. By using the 3-step purificationprocedure, approximately 16 mg of the purified material from 1 liter ofsupernatant was obtained. The starting supernatants had different levelsof immunotoxin aggregates and monomeric immunotoxin depending on theinduction time during fermentation run. Among these immunotoxinaggregates, some portions could be reversible to monomeric form of theimmunotoxin during the Butyl 650M HIC step. Fractionation of supernatantby gel filtration and subsequent SDS-PAGE analysis showed that thesupernatants contained more than 50% of the immunotoxin aggregates.However, the final yield of the immunotoxin after the 3-steppurification procedure was 52.8%, indicating that a portion of theaggregates could be dissociated into monomeric immunotoxin duringpurification.

A comparison of the purification procedure applied to 3 separatefermentation runs that contained similar amounts of supernatantimmunotoxin demonstrates good repeatability of the procedure withrespect to yields (Table 5).

The purity of the purified immunotoxin was assessed by analytical gelfiltration. The immunotoxin in the final preparation displayed a singlepeak corresponding to the monomeric form of the immunotoxin (panel A inFIG. 19). The analyses of purity of the final preparations confirmedthat the 3-step purification yielded an immunotoxin with ˜98.0% purity(panel B in FIG. 19).

To investigate the effects of the 3-step purification procedure onimmunotoxin bioactivity, a protein synthesis assay for the specific Tcell toxicity of the final preparation was performed. The estimatedconcentration of the immunotoxin in the final preparation coincided withconcentration of the immunotoxin standard.

TABLE 5 Comparison of immunotoxin purification from Pichia pastorisfermentor cultures*. Batch IT conc volume total IT yield acc. yield no.step (ug/ml) (ml) (mg) (%) (%) 1 Supernatant 30.0 1000 30.0 100.0 100.0Butyl 650M 45.0 585 26.3 87.8 87.8 HIC Poros 50 HQ 15.0 1200 18.0 67.060.0 borate AEX Q AEX 400.0 40 16.0 88.9 53.3 2 Supernatant 30.0 100030.0 100.0 100.0 Butyl 650M 40.0 585 23.4 78.0 78.0 HIC Poros 50 HQ 15.01200 17.5 74.6 58.2 borate AEX Q AEX 400.0 40 16.0 91.6 53.3 3Supernatant 30.0 1000 30.0 100.0 100.0 Butyl 650M 40.0 585 23.4 78.078.0 HIC Poros 50 HQ 15.0 1200 18.0 67.0 60.0 borate AEX Q AEX 450.0 3515.8 87.5 52.5 *IT, immunotoxin; acc., accumulated; HIC, hydrophobicinteraction chromatography; AEX, anion exchange chromatography.Supernatants were obtained from 3 fermentation runs at 44 hours ofmethanol induction.

Example 40 Summary

Glycerol feeding decreased immunotoxin proteolysis and enhancedimmunotoxin production while yeast extract feeding primarily enhancedmethanol utilization and cell growth. Glycerol feeding and yeast extractfeeding acted synergistically to increase immunotoxin production andthis synergy was enhanced at 15° C.

This study demonstrates a synergy between carbon source supplementationwith glycerol and continuous yeast extract feeding that attenuates thetoxic effects of the immunotoxin and increases production, especially at15° C. This robust process has a yield of 37 mg/L, 7-fold greater thanthat previously reported in the toxin-resistant CHO cell expressionsystem (25).

Example 41

The final method uses the toxin resistant EF-2 mutant, limited methanolfeeding during induction of 0.5 to 0.75 ml/min (per 10 L initial medium)without an additional carbon source, extension of induction time to 163h, a temperature of 15° C., a continuous infusion of yeast extract,limitation of agitation speed to 400 RPM, addition of antifoam agent upto 0.07%, and supplementation of oxygen when DO levels fall below 40%.Under these conditions PMSF and Casamino acids are not required.Additionally, reducing shearing force by lowering agitation speed andadding anti-foam reagent dramatically reduced immunotoxin aggregation.As a result, purification yield was improved from 64 to 76%. Under thisoptimized methodology, immunotoxin secretion level was 120 mg/L at 163hr of methanol induction. Table 6 summarizes how much immunotoxinsecretion and purification yield were improved by solving the majorproblems. Gene optimization enhanced IT secretion from non-detectablelevel to 10 mg/L. By using DT-resistant strain and employing lowtemperature, we improved immunotoxin secretion up to 35 mg/L.Furthermore, employing limited methanol feeding improved immunotoxinsecretion as well as purification yield. Finally, extension of inductiontime and addition of anti-foam reagent dramatically increasedimmunotoxin secretion and purification yield. The anti-foam reagent isKFOTM 673 which was purchased from Kabo Chemical, Inc. (Cheyennne, Wyo.82007, USA).This methodology can be useful for the production of otherrecombinant immunotoxins and other toxic proteins in toxin-sensitive P.pastoris.

TABLE 6 Increase in immunotoxin secretion and purification yield bysolving major problems IT secretion solutions level Purification yieldGene optimization 10.0 mg/L n.a. Use of DT-resistant strain 35.0 mg/L14.5 mg/L (41.4%) & low temperature Limited methanol feeding 50.0 mg/L32.0 mg/L (64.0%) Extended induction time & 120.0 mg/L  90.8 mg/L(75.7%) addition of anti-foam reagent

Throughout this application various publications are referenced. Fullcitations for these publications are as follow. Such publicationsmentioned are hereby incorporated in their entirety by reference inorder to more fully describe the state of the art to which thisinvention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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1-26. (canceled)
 27. A method of purifying a non-glycosylatedimmunotoxin comprising a) loading a solution containing thenon-glycosylated immunotoxin onto a hydrophobic interaction column; b)obtaining a first non-glycosylated immunotoxin containing eluant fromthe hydrophobic interaction column; c) loading the non-glycosylatedimmunotoxin containing eluant from step (b) onto an anion exchangecolumn; d) obtaining a second non-glycosylated immunotoxin containingeluant from the anion exchange column by eluting the non-glycosylatedimmunotoxin with a sodium borate solution; e) diluting the concentrationof sodium borate in the second non-glycosylated immunotoxin containingeluant from step (d) to about 50 ruM or less; f) concentrating thediluted non-glycosylated immunotoxin containing eluant from step (e)over an anion exchange column; and g) obtaining a purifiednon-glycosylated immunotoxin from the anion exchange column.
 28. Themethod of claim 27, wherein the non-glycosylated immunotoxin isexpressed in yeast.
 29. The method of claim 28, wherein the yeast isPichia pastoris.
 30. The method of claim 27, wherein the immunotoxin isa fusion protein.
 31. The method of claim 27, wherein the immunotoxincomprises a diphtheria toxin moiety.
 32. The method of claim 31, whereinthe diphtheria toxin moiety is truncated.
 33. The method of claim 32,farther comprising a CD3 antibody moiety.
 34. The method of claim 33,wherein the non-glycosylated immunotoxin comprises A-dmDT390-bisFv(G4S).35. The method of claim 27, further comprising washing the anionexchange column with about 25 mM sodium borate solution prior to elutingwith the sodium borate solution.
 36. The method of claim 27, wherein theconcentration of the sodium borate solution in step (d) is between about50 mM and about 200 mM.
 37. The method of claim 36, wherein theconcentration of the sodium borate solution in step (d) is between about75 mM and about 100 mM.
 38. The method of claim 27, wherein theconcentration of sodium borate in step (e) is about 20 mM.